Prodrug compounds with isoleucine

ABSTRACT

The compounds of the invention are modified forms of therapeutic agents. A typical prodrug compound of the invention comprises a therapeutic agent, an oligopeptide having an isoleucine residue, a stabilizing group and, optionally, a linker group. The prodrug is cleavable by an enzyme associated with the target cell. Methods of making and using the compounds are also disclosed.

RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.11/540,466, filed Sep. 29, 2006, issuing; which is a divisionalapplication of U.S. Ser. No. 10/311,411, filed Dec. 13, 2002, now U.S.Pat. No. 7,115,573, issued Oct. 3, 2006; which is a 35 U.S.C. §371application of PCT/US01/18857, filed Jun. 11, 2001; which claimspriority to U.S. Ser. No. 60/211,686, filed Jun. 14, 2000. The contentsof all of the aforementioned applications are hereby incorporated byreference in their entirety.

TECHNICAL FIELD

The present invention is directed to new compounds useful as prodrugs.Such prodrugs may be used for treating disease, especially tumors, inpatients.

BACKGROUND

Many therapeutic agents, such as anthracyclines and vinca alkaloids, areespecially effective for the treatment of cancers. However, thesemolecules are often characterized in vivo by an acute toxicity,especially a bone marrow and mucosal toxicity, as well as a chroniccardiac toxicity in the case of the anthracyclines and chronicneurological toxicity in the case of the vinca alkaloids. Similarly,methotrexate may be used for the treatment of inflammatory reactions,such as rheumatic diseases, but its high toxicity limits itsapplications. Development of more specific and safer antitumor agents isdesirable for greater effectiveness against tumor cells and a decreasein the number and severity of the side effects of these products(toxicity, destruction of non-tumor cells, etc.). Development of morespecific anti-inflammatory agents is also desirable.

In order to minimize toxicity problems, therapeutic agents areadvantageously presented to patients in the form of prodrugs. Prodrugsare molecules capable of being converted to drugs (active therapeuticcompounds) in vivo by certain chemical or enzymatic modifications oftheir structure. For purposes of reducing toxicity, this conversionshould be confined to the site of action or target tissue rather thanthe circulatory system or non-target tissue. Prodrugs are oftencharacterized by a low stability in blood and serum, however. This isdue to the presence of enzymes in blood and serum that degrade, andconsequently may activate, the prodrugs before the prodrugs can reachthe desired sites within the patient's body.

A desirable class of prodrugs that overcomes such problems have beendisclosed in Patent Cooperation Treaty International Publication No. WO96/05863 and in U.S. Pat. No. 5,962,216, both incorporated herein byreference. Further useful prodrug compounds and methods of making suchprodrugs are desirable, however, as are methods of making the prodrugs.

Prodrugs that display a high specificity of action, a reduced toxicity,and an improved stability in blood especially relative to prodrugs ofsimilar structure that have existed in the public domain areparticularly desirable.

SUMMARY OF THE INVENTION

The compound of the invention is a prodrug form of a therapeutic agent,in which the therapeutic agent is linked directly or indirectly to anoligopeptide, which in turn, is linked to a stabilizing group. Theoligopeptide has an isoleucine residue in the third amino acid positioncounted from the C-terminus (per the typical orientation of theprodrug). The prodrugs of the invention display a high specificity ofaction, a reduced toxicity, an improved stability in the serum andblood, and do not move into target cells, or do so only minimally, untilactivated by a target cell associated enzyme.

The present invention also relates to the pharmaceutical compositioncomprising the compound according to the invention and optionally apharmaceutically acceptable carrier, adjuvant, vehicle or the like.Articles of manufacture, such as kits for diagnosis or assay are alsodescribed.

Further, methods of designing prodrugs and of decreasing toxicity animproving safety index by modifying a therapeutic agent to create aprodrug are disclosed. Such modification provides an improvedtherapeutic index as compared to the free therapeutic agent.

The present invention further includes methods of treating a medicalcondition by administering the prodrug of the invention.

Several processes for making the prodrugs are included, as well.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D are a table of abbreviations, names, and structures

FIG. 2 is an exemplary scheme of cleavage of a prodrug of the inventionin the extracellular vicinity of the target cell and within the targetcell.

FIG. 3 illustrates a synthesis of Fmoc-βAla-Ile-Ala-Leu (SEQ ID NO:37),a typical intermediate of the invention.

FIG. 4 illustrates an “Fmoc-route” synthesis ofMethyl-succinyl-βAla-Ile-Ala-Leu (SEQ ID NO:38), a typical intermediateof the invention.

FIG. 5 illustrates an “Fmoc route” synthesis of a salt form ofSuc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:39), a typical compound of theinvention.

FIG. 6 illustrates an “Ester route” synthesis of a salt form ofSuc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:39), a typical compound of theinvention.

FIG. 7 illustrates a synthesis of an amino-protectedβAla-Ile-Ala-Leu-Dox (SEQ ID NO:40), a typical intermediate of theinvention.

FIG. 8 illustrates an “Allyl ester route” synthesis of a salt form ofSuc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:39), a typical compound of theinvention.

FIG. 9 illustrates a “Resin route” synthesis of Suc-βAla-Ile-Ala-Leu-Dox(SEQ ID NO:39), a typical compound of the invention.

FIG. 10 is a table of oligopeptides useful in the prodrug of theinvention.

FIG. 11 is a graph of the plasma levels of Suc-Ile-Ala-Leu-Dox and itsmetabolites at 1 and 4 hours after administration of a singleintravenous bolus dose of the prodrug.

FIG. 12 is a graph of the amount of Suc-Ile-Ala-Leu-Dox and itsmetabolites present in the urine collected 0-2 and 2-24 hours after theadministration of a single intravenous bolus of the prodrug.

FIG. 13 is a graph of the plasma levels of Suc-βAla-Ile-Ala-Leu-Dox (SEQID NO:39) and its metabolites at 1 and 4 hours after administration of asingle intravenous bolus dose of the prodrug.

FIG. 14 is a graph of the amount of Suc-βAla-Ile-Ala-Leu-Dox (SEQ IDNO:39) and its metabolites present in the urine collected 0-2 and 2-24hours after the administration of a single intravenous bolus of theprodrug.

FIG. 15 is a graph of the Percent Body Weight Change of either micetreated with Suc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:39) or mice receivingthe vehicle control.

FIG. 16 is a graph of the rate of tumor growth in LS174T xenograftedmice either treated with Suc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:39) orgiven the vehicle control.

FIG. 17 illustrates the removal of free therapeutic agent through theuse of scavenging resin or beads.

DETAILED DESCRIPTION Abbreviations

ACN=Acetonitrile

Aib=Aminoisobutyric acid

All=Allyl

Aloc=Allyloxycarbonyl

Amb=4-(Aminomethyl)benzoic acid

APP=3-Amino-3-phenylpropionic acid

DCC=N,N′-Dicyclohexylcarbodiimide

Boc=t-butyloxycarbonyl

Cap=amino caproic acid

DBN=1,5 Diazabicyclo[4.3.0]non-5-ene

DBO=1,4 Diazabicyclo[2.2.2]octane

DBU=1,8-Diazabicyclo[5.4.0]undec-7-ene

DCM=Dichloromethane

DIC=N,N′-Diisopropylcarbodiimide

DIEA=Diisopropylethylamine

Dg=Diglycolic Acid

DMF=Dimethylformamide

Dnr=Daunorubicin

Dox=Doxorubicin

Et₂O=diethyl ether

Fmoc=9-Fluorenylmethyloxycarbonyl

GI=Glutaric Acid

HATU=O-(7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium-hexafluorophosphate

HBTU=2-(1H-Benzotriazole-1-yl)1,1,3,3-tetramethyluronium-hexafluorophosphate

HEPES=Hydroxethylpiperidine

HOBt=N-Hydroxybenzotriazole

HPLC=High pressure liquid chromatography

MeOH=Methanol

MeOSuc=Methyl hemisuccinyl/Methyl hemisuccinate

MTD=Maximum tolerated dose

NAA=3-Amino-4,4-diphenylbutyric Acid

Nal=2-Naphthylalanine

Naph=1,8-Naphthalene dicarboxylic acid

Nle=Norleucine

NMP=N-methylpyrrolidine

Nva=Norvaline

PAM resin=4-hydroxymethylphenylacetamidomethyl

Pyg=Pyroglutamic acid

Pyr=3-Pyridylalanine

RT, rt=Room temperature

Suc=Succinic Acid/Succinyl

TCE=trichloroethyl

TFA=Trifluoroacetic acid

THF=Tetrahydrofuran

Thi=2-Thienylalanine

Thz=Thiazolidine-4-carboxylic acid

Tic=Tetrahydroisoquinoline-3-carboxylic acid

TOP=Thimet oligopeptidase

The invention includes compounds that may be described as prodrug formsof therapeutic agents. The therapeutic agent is linked directly orindirectly to an oligopeptide, which in turn, is linked to a stabilizinggroup. A linker group between the therapeutic agent and the oligopeptidemay optionally be present. The oligopeptide is three or four amino acidsin length and is characterized, in the typical orientation of theprodrug, by having an isoleucine residue in the third amino acid fromits C-terminus.

Prodrug

The prodrug of the invention is a modified form of a therapeutic agentand comprises several portions, including:

(1) a therapeutic agent,

(2) an oligopeptide, and

(3) a stabilizing group, and

(4) optionally, a linker group.

Each of the portions of the prodrug are discussed in greater detailbelow. The typical orientation of these portions of the prodrug is asfollows:

(stabilizing group)-(oligopeptide)-(optional linker group)-(therapeuticagent).

The stabilizing group is directly linked to the oligopeptide at a firstattachment site of the oligopeptide. The oligopeptide is directly orindirectly linked to the therapeutic agent at a second attachment siteof the oligopeptide. If the oligopeptide and the therapeutic agent areindirectly linked, then a linker group is present.

Direct linkage of two portions of the prodrug means a covalent bondexists between the two portions. The stabilizing group and theoligopeptide are therefore directly linked via a covalent chemical bondat the first attachment site of the oligopeptide, typically theN-terminus of the oligopeptide. When the oligopeptide and thetherapeutic agent are directly linked then they are covalently bound toone another at the second attachment site of the oligopeptide. Thesecond attachment site of the oligopeptide is typically the C-terminusof the oligopeptide, but may be elsewhere on the oligopeptide.

Indirect linkage of two portions of the prodrug means each of the twoportions is covalently bound to a linker group. In an alternativeembodiment, the prodrug has indirect linkage of the oligopeptide to thetherapeutic agent. Thus, typically, the oligopeptide is covalently boundto the linker group which, in turn, is covalently bound to thetherapeutic agent.

The prodrug of the invention is typically cleavable within itsoligopeptide portion. In order for the prodrug to be effective, theprodrug typically undergoes in vivo modification producing a portion ofthe prodrug that is able to enter the target cell. A first cleavagewithin the oligopeptide portion of the prodrug may leave an activeportion of the prodrug, i.e., a portion of the prodrug that is competentfor transport into the target cell, as one of the cleavage products.Alternatively, further cleavage by one or more peptidases may berequired to result in a transport-competent portion of the prodrug. Theactive or transport-competent portion of the prodrug has at least thetherapeutic agent and is that part of the prodrug that can enter thetarget cell to exert a therapeutic effect directly or upon furtherconversion within the target cell.

Thus, the compound has an active portion, and the active portion is morecapable of entering the target cell after cleavage by an enzymeassociated with a target cell than prior to such cleavage. Thestructures of the stabilizing group and oligopeptide are selected tolimit clearance and metabolism of the prodrug by enzymes, which may bepresent in blood or non-target tissues and are further selected to limittransport of the prodrug into cells. The stabilizing group blockscleavage of the prodrug by exopeptidase in vivo and, additionally, mayact in providing preferable charge or other physical characteristics ofthe prodrug. The amino acid sequence of the oligopeptide is selected forresistance to cleavage by trouase, a class of enzymes associated withtarget cells and described in greater detail below. Both tetrapeptidesand tripeptides may be used in the invention. In a preferred embodimentof the invention, the isoleucine-containing peptide is a tripeptide.Generally speaking, tripeptides are poorly cleaved in the systemiccirculation and in normal tissues and therefore may have a highertherapeutic index as compared with a tetrapeptide of overlappingsequence.

It is desirable to make a therapeutic agent, especially an antitumorand/or anti-inflammatory therapeutic agent, inactive by modification ofthe therapeutic agent to a prodrug form. According to the invention, thetarget cells are usually tumor cells or cells, such as macrophages,neutrophils, and monocytes, participating in inflammatory reactions,especially those associated with rheumatic diseases. Modification of thetherapeutic agent to a prodrug form also reduces some of the sideeffects of the therapeutic agents.

The prodrug is administered to the patient, carried through the bloodstream in a stable form, and when in the vicinity of a target cell, isrecognized and modified by a target cell associated enzyme. Since theenzyme activity is only minimally present within the extracellularvicinity of normal cells, the prodrug is not activated and itstransport-competent portion (including the therapeutic agent) gainsentry into the normal cells only minimally at best. In the vicinity oftumor or other target cells, however, the increased presence of therelevant enzyme in the local environment causes cleavage of the prodrug.After modification from the prodrug form and entry into the target cell,the therapeutic agent (optionally attached to one or more amino acidsand possibly also a linker group) acts to kill or block proliferation ofthe target cell. The example shown in FIG. 2 depicts a typical prodrugbeing cleaved extracellularly and gaining entry into the target cell.Once within the target cell, it may be further modified to providetherapeutic effect. While a portion of the prodrug may occasionally gainaccess to and possibly harm normal cells, the transport-competentportion of the prodrug is freed primarily in the vicinity of targetcells. Thus, toxicity to normal cells is minimized.

This process is particularly useful for, and is designed for, targetcell destruction when the target tissue releases an enzyme that is notreleased by normal tissue or cells. Here “normal cells” means non-targetcells that would be encountered by the prodrug upon administration ofthe prodrug in the manner appropriate for its intended use.

In an alternative embodiment, the orientation of the prodrug may bereversed so that a stabilizing group is attached to the oligopeptide atthe C-terminus and the therapeutic agent is directly or indirectlylinked to the N-terminus of the oligopeptide. Thus, in an alternativeembodiment, the first attachment site of the oligopeptide may be theC-terminus of the oligopeptide and the second attachment site by theoligopeptide may be the N-terminus of the oligopeptide. The linker groupmay optimally be present between the therapeutic agent and theoligopeptide. The alternative embodiment of the prodrug of the inventionfunctions in the same manner as does the primary embodiment.

It was expected that oligopeptide sequences having isoleucine in the AA³position (per the numbering scheme described below) would not make goodcandidates for prodrugs due to the preferential cleavage by trouase ofoligopeptides having a non-isoleucine residue in the AA³ position.However, when isoleucine-containing prodrug compounds, as describedherein, were tested in vivo, the surprising discovery was that suchcompounds did indeed serve as useful prodrugs.

Compounds of the invention are good prodrugs because they show highstability, i.e., a low level of release of the active therapeutic agent,in the systemic circulation and in normal tissues. Peptides of theinvention are not activated in the blood or normal tissues to any greatextent, yet are activated at tumor sites. Consequently, they have animproved therapeutic index, improved toxicological profile and favorablepharmacokinetics.

Without limitation to a particular theory, it is believed that suchisoleucine-containing prodrugs are activated in the vicinity of thetarget cells by an enzyme other than a trouase-type enzyme. Thus,creation of such isoleucine-containing prodrugs represents analternative method of prodrug design to that presented inPCT/US99/30393.

As described herein, a compound of the invention comprises:

(1) a therapeutic agent capable of entering a target cell,

(2) an oligopeptide of the formula (AA)_(n)-AA³-AA²-AA¹, wherein:

-   -   each AA independently represents an amino acid,    -   n is 0 or 1, and when n is 1, then (AA)_(n) is AA⁴ which        represents any amino acid,    -   AA³ represents isoleucine,    -   AA² represents any amino acid, and    -   AA¹ represents any amino acid,

(3) a stabilizing group, and

(4) optionally, a linker group not cleavable by a trouase, such as TOP(described in greater detail below)

wherein the oligopeptide is directly linked to the stabilizing group ata first attachment site of the oligopeptide and the oligopeptide isdirectly linked to the therapeutic agent or indirectly linked throughthe linker group to the therapeutic agent at a second attachment site ofthe oligopeptide,

wherein the stabilizing group hinders cleavage of the compound byenzymes present in whole blood, and

wherein the compound is cleavable by an enzyme associated with thetarget cell, the enzyme associated with the target cell being other thanTOP. The compound preferably includes an oligopeptide that is resistantto cleavage by a trouase, particularly TOP i.e., resistant to cleavageunder physiological conditions. The optionally present linker group thatis not cleavable by a trouase is not cleavable under physiologicalconditions.

For purposes of this discussion, a compound is resistant to cleavage bya given enzyme if the rate of cleavage by a purified preparation of thegiven enzyme is no more than 15%, preferably no more than 5%, andideally no more than 1% of the rate of cleavage of Suc-βAla-Leu-Ala-Leu(SEQ ID NO:41) conjugated via the carboxyl terminus to the sametherapeutic agent as the compound of interest. The rates should becompared under the same assay conditions.

A compound is cleavable by a given enzyme if greater than 10% per hour,preferably greater than 50% per hour, is cleaved by a mixture of thecompound and the enzyme under experimental conditions which modelphysiological conditions, particularly those outside of the target cell.The concentration of the given enzyme in the experiment isrepresentative of the concentration of the given enzyme in theextracellular milieu of the target tissue.

Target Cell Associated Enzymes

The prodrugs of the invention are designed to take advantage ofpreferential activation, through interaction with an enzyme associatedwith the target cell, at or near the site targeted within the body ofthe patient. Although not believed to be responsible for cleavage of thecompounds of the invention, one such type of enzyme, or class ofenzymes, associated with likely target cells is trouase, described ingreater detail in PCT/US99/3.0393. Trouase is believed to be a class ofenzymes, of which Thimet oligopeptidase (“TOP”) is one member. Trouasesare highly discriminating in their selectivity and cleavage. Trouase isan endopeptidase that shows a remarkable degree of discriminationbetween leucine and isoleucine at the carboxyl side of the oligopeptidecleavage site. A defining characteristic is that under appropriate assayconditions, trouase readily cleavessuccinyl-βAla-Leu-Ala-Leu-Daunorubicin (SEQ ID NO:42) while it is atleast twenty-fold less active withsuccinyl-βAla-Ile-Ala-Leu-Daunorubicin (SEQ ID NO:43).

Although knowledge of the sequences cleaved by trouase may be utilizedfor designing therapeutically useful prodrugs, the previouslyless-favored isoleucine-containing peptide sequences represent anunexpectedly useful alternative for designing prodrugs. The presence ofisoleucine at the P1 cleavage site of trouase (which would be equivalentto the AA³ position in the typical oligopeptides described herein) hasbeen shown to prevent or greatly minimize cleavage of the peptide bytrouase. Such inhibition was shown both in vitro using partiallypurified trouase and purified TOP and in vivo in metabolic studies withnormal mice.

The enzyme involved in the activation of the prodrugs of the inventionis believed to be associated with target cells, but is found in thecirculation only at very low levels. Most likely it is generated eitherby the target cells themselves or by normal cells that are associatedwith the target cells, such as stromal cells, neutrophils, ormacrophages, or B cells. So, for example, the target cell associatedenzyme may be associated with or bound on (at least the active site) theouter cell surface, secreted, released, or present in some other mannerin the extracellular vicinity of the target cell. In many cases, theprodrug of the invention includes a therapeutic agent for the treatmentof cancer and the target cell is a tumor cell. Thus, the enzyme may besecreted extracellularly by the tumor cell or it may be presentextracellularly because there is a fair amount of cell lysis associatedwith tumors generally.

Despite lack of cleavage by trouase and especially the TOP enzyme, invivo studies have shown that compounds with isoleucine in what would bethe trouase P1 cleavage position are effective in reducing the tumorgrowth rate and extending survival of nude mice implanted with humantumor xenografts. This finding indicates that one or more enzymes arepresent in tumor-bearing animals which can cleave isoleucine-containingprodrug compounds to initiate a process eventually leading to release ofthe active portion of the therapeutic agent, usually a cytotoxic agent,within the tumor or other target cells. The enzyme or enzymes thatcleave isoleucine-containing peptides may be even more tumor-specificthan TOP, which is present in a wide variety of normal cells and celltypes as well as human cancer cells. Thus, designing of prodrugs havingan isoleucine at an important position within the oligopeptide portionof the prodrug may be preferable to previously disclosed compounds thatare cleavable by trouase and/or TOP.

Stabilizing Group

An important portion of the prodrug is the stabilizing group, whichserves to protect the prodrug compound from cleavage in circulatingblood when it is administered to the patient and allows the prodrug toreach the vicinity of the target cell relatively intact. The stabilizinggroup typically protects the prodrug from cleavage by proteinases andpeptidases present in blood, blood serum, and normal tissue.Particularly, since the stabilizing group caps the N-terminus of theoligopeptide, and is therefore sometimes referred to as an N-cap orN-block, it serves to ward against peptidases to which the prodrug mayotherwise be susceptible.

Ideally, the stabilizing group is useful in the prodrug of the inventionif it serves to protect the prodrug from degradation, i.e., cleavage,when tested by storage of the prodrug compound in human blood at 37° C.for 2 hours and results in less than 20%, preferably less than 2%,cleavage of the prodrug by the enzymes present in the human blood underthe given assay conditions. Thus, a fully formed conjugate may be testedto see if it is stable in whole blood.

More particularly, a stabilizing group that hinders cleavage of theoligopeptide by enzymes present in whole blood is advantageously chosenfrom the following:

(1) other than an amino acid, or

(2) an amino acid that is either (i) a non-genetically-encoded aminoacid or (ii) aspartic acid or glutamic acid attached to the N-terminusof the oligopeptide at the β-carboxyl group of aspartic acid or theγ-carboxyl group of glutamic acid.

For example, dicarboxylic (or a higher order carboxylic) acid or apharmaceutically acceptable salt thereof may be used as a stabilizinggroup. Since chemical radicals having more than two carboxylic acids arealso acceptable as part of the prodrug, the end group havingdicarboxylic (or higher order carboxylic) acids is an exemplary N-cap.The N-cap may thus be a monoamide derivative of a chemical radicalcontaining two or more carboxylic acids where the amide is attached ontothe amino terminus of the peptide and the remaining carboxylic acids arefree and uncoupled. For this purpose, the N-cap is preferably succinicacid, adipic acid, glutaric acid, or phthalic acid, with succinic acidand adipic acid being most preferred. Other examples of useful N-caps inthe prodrug compound of the invention include diglycolic acid, fumaricacid, naphthalene dicarboxylic acid, pyroglutamic acid, acetic acid, 1-or 2-, naphthylcarboxylic acid, 1,8-naphthyl dicarboxylic acid, aconiticacid, carboxycinnamic acid, triazole dicarboxylic acid, gluconic acid,4-carboxyphenyl boronic acid, a (PEG)_(n)-analog such as polyethyleneglycolic acid, butane disulfonic acid, maleic acid, nipecotic acid, andisonipecotic acid.

Further, a non-genetically encoded amino acid such as one of thefollowing may also be used as the stabilizing group: β-Alanine,Thiazolidine-4-carboxylic acid, 2-Thienylalanine, 2-Naphthylalanine,D-Alanine, D-Leucine, D-Methionine, D-Phenylalanine,3-Amino-3-phenylpropionic acid, γ-Aminobutyric acid,3-amino-4,4-diphenylbutyric acid, Tetrahydroisoquinoline-3-carboxylicacid, 4-Aminomethylbenzoic acid, and Aminoisobutyric acid.

Additionally, in some experiments intravascular administration of anaggregating positively charged prodrug in mice resulted in acutetoxicity. However, no such toxicity was observed when the charge on thisprodrug was reversed by derivitization with a negatively chargedstabilizing group.

Many cytotoxic compounds inherently have low solubility. Positivelycharged anthracyclines for example may form aggregates at highconcentration and these aggregates may induce intravenous coagulationwhen the aggregates are administered intravenously. Since manyoligopeptides have exposed, positively-charged amino termini atphysiological pH, these aggregates may form a polypositively chargedsurface in vivo and induce a coagulation cascade within a few minutes ofadministration. This has the potential for rendering any positivelycharged prodrugs that form aggregates unsuitable for therapeutic use.

As described in greater detail in PCT/US99/30393, one way of addressingsuch a potentially dangerous obstacle is to utilize the stabilizinggroup on the peptide chain N-terminus of a negatively charged or aneutral functionality. For example, the use of succinyl as a stabilizinggroup on the prodrug alleviates the prodrug's acute toxicity. It isbelieved that the stabilizing group reduces interaction between thecompound and endothelial cells that line blood vessels. This solves animportant problem in the use of peptide prodrugs as practical therapiesfor intravenous use in humans.

Oligopeptide

Oligopeptides are generally defined as polypeptides of short length,typically twenty amino acids or fewer. An oligopeptide useful in theprodrug of the invention is three or four amino acids in length.However, oligopeptides of greater length are also possible.

Numbering Scheme

According to the invention, the oligopeptide portion of the prodrug hasa formula (AA)_(n)-AA³-AA²-AA¹, wherein:

each AA independently represents an amino acid,

n is 0 or 1, and when n is 1, then (AA)_(n) is AA⁴ which represents anyamino acid,

AA³ represents isoleucine,

AA² represents any amino acid, and

AA¹ represents any amino acid.

The oligopeptide is written in the conventional manner with the carboxylterminus at the right and the amino terminus at the left. It is believedthat the enzyme associated with the target cell cleaves the linkagebetween AA¹ and AA² of the oligopeptide. Unless otherwise indicated, allamino acids are in the L configuration. Although any amino acids may bepresent in the oligopeptide portion of the prodrug, with the exceptionof the amino acid at AA³, which is always isoleucine, certain aminoacids are preferred.

In the AA⁴ position of the oligopeptide portion, a non-geneticallyencoded amino acid, for example, one of the following amino acids, ismost preferably present: β-Alanine, Thiazolidine-4-carboxylic acid,2-Thienylalanine, 2-Naphthylalanine, Alanine, D-Alanine, D-Leucine,D-Methionine, D-Phenylalanine, 3-Amino-3-phenylpropionic acid,3-amino-4,4-diphenylbutyric acid, or Proline. Also possible areAminoisobutyric acid, 4-Aminomethylbenzoic acid, orTetrahydroisoquinoline-3-carboxylic acid in the AA⁴ position.

AA³ of the oligopeptide portion of the prodrug of the invention isisoleucine.

Most preferably, in the AA² position of the oligopeptide portion of theprodrug is one of the following amino acids: Alanine, Leucine, Glycine,Serine, Tyrosine, 3-Pyridylalanine, 2-Thienylalanine, orN-Methyl-alanine. The amino acid in the AA² position may also beselected from Aminoisobutyric Acid, Threonine, or Phenylalanine.

The amino acid present in the AA¹ position is most preferably selectedfrom one of the following: Leucine, Phenylalanine, Isoleucine, Alanine,Glycine, Tyrosine, 2-Naphthylalanine, Serine, or Proline. Also preferredin the AA¹ position is β-Alanine.

Oligopeptides useful in the prodrug of the invention include those shownin FIG. 10, particularly βAla-Ile-Ala-Phe (SEQ ID NO: 1);βAla-Ile-Ala-Ile (SEQ ID NO: 2); Tic-Ile-Ala-Leu (SEQ ID NO: 3);Thi-Ile-Ala-Leu (SEQ ID NO: 4); Nal-Ile-Ala-Leu (SEQ ID NO: 5);Amb-Ile-Ala-Leu (SEQ ID NO: 6); Aib-Ile-Ala-Leu (SEQ ID NO: 7);βAla-Ile-Ala-Leu (SEQ ID NO: 8); Thi-Ile-Aib-Leu (SEQ ID NO: 9);Nal-Ile-Aib-Leu (SEQ ID NO: 10); βAla-Ile-Aib-Leu (SEQ ID NO: 11);Amb-Ile-Aib-Leu (SEQ ID NO: 12); Aib-Ile-Aib-Leu (SEQ ID NO: 13);βAla-Ile-Gly-Phe (SEQ ID NO: 14); βAla-Ile-Gly-Ile (SEQ ID NO: 15);Tic-Ile-Gly-Leu (SEQ ID NO: 16); Thi-Ile-Gly-Leu (SEQ ID NO: 17);Nal-Ile-Gly-Leu (SEQ ID NO: 18); βAla-Ile-Gly-Leu (SEQ ID NO: 19);Amb-Ile-Gly-Leu (SEQ ID NO: 20); Aib-Ile-Gly-Leu (SEQ ID NO: 21);βAla-Ile-Thr-Ile (SEQ ID NO: 22); βAla-Ile-Tyr-Ile (SEQ ID NO: 23), andβAla-Ile-Ala-Gly (SEQ ID NO: 24). Preferred tripeptide sequences for theinvention include Ile-Ala-Leu (SEQ ID NO: 25); Ile-N(Me)Ala-Leu (SEQ IDNO: 26); Ile-Ala-Phe (SEQ ID NO: 27); Ile-Ala-Ile (SEQ ID NO: 28);Ile-Aib-Leu (SEQ ID NO: 29); Ile-Gly-Phe (SEQ ID NO: 30); Ile-Gly-Ile(SEQ ID NO: 31); Ile-Gly-Leu (SEQ ID NO: 32); Ile-Thr-Ile (SEQ ID NO:33); and Ile-Ala-Gly (SEQ ID NO: 34); βAla-Ile-Tyr-Leu (SEQ ID NO: 35);and βAla-Ile-Tyr-Gly (SEQ ID NO: 36).

Therapeutic Agents

Therapeutic agents that are particularly advantageous to modify to aprodrug form are those with a narrow therapeutic window. A drug ortherapeutic agent with a narrow therapeutic window is one in which thedose at which toxicity is evident, by general medical standards, is veryclose to the dose at which efficacy is evident.

The therapeutic agent conjugated to the stabilizing group andoligopeptide and, optionally, the linker group to form the prodrug ofthe invention may be useful for treatment of cancer, inflammatorydisease, or some other medical condition. Preferably, the therapeuticagent is selected from the following classes of compounds: AlkylatingAgents, Antiproliferative agents, Tubulin Binding agents, VincaAlkaloids, Enediynes, Podophyllotoxins or Podophyllotoxin derivatives,the Pteridine family of drugs, Taxanes, Anthracyclines, Dolastatins,Topoiosomerase inhibitors, Platinum-coordination-complexchemotherapeutic agents, Maytansinoids.

Particularly, the therapeutic agent is advantageously selected from thefollowing compounds, or a derivative or analog thereof: Doxorubicin,Daunorubicin, Vinblastine, Vincristine, Calicheamicin, Etoposide,Etoposide phosphate, CC-1065, Duocarmycin, KW-2189, Methotrexate,Methopterin, Aminopterin, Dichloromethotrexate, Docetaxel, Paclitaxel,Epithiolone, Combretastatin, Combretastatin A4 Phosphate, Dolastatin 10,Dolastatin 11, Dolastatin 15, Topotecan, Camptothecin, Mitomycin C,Porfiromycin, 5-Fluorouracil, 6-Mercaptopurine, Fludarabine, Tamoxifen,Cytosine arabinoside, Adenosine Arabinoside, Colchicine, Cisplatin,Carboplatin, Mitomycin C, Bleomycin, Melphalan, chloroquine, cyclosporinA, and Maytansine. By derivative is intended a compound that resultsfrom reacting the named compound with another chemical moiety, andincludes a pharmaceutically acceptable salt, acid, base or ester of thenamed compound. By analog is intended a compound having similarstructural and functional properties, such as biological activities, tothe named compound.

Linker Groups

A linker group between the oligopeptide and the therapeutic agent may beadvantageous for reasons such as the following:

-   -   1. As a spacer for steric considerations in order to facilitate        enzymatic release of the AA¹ amino acid or other enzymatic        activation steps.    -   2. To provide an appropriate attachment chemistry between the        therapeutic agent and the oligopeptide.    -   3. To improve the synthetic process of making the prodrug        conjugate (e.g., by pre-derivitizing the therapeutic agent or        oligopeptide with the linker group before conjugation to enhance        yield or specificity.)    -   4. To improve physical properties of the prodrug.    -   5. To provide an additional mechanism for intracellular release        of the drug.

Linker structures are dictated by the required functionality. Examplesof potential linker chemistries are hydrazide, ester, ether, andsulfhydryl. Amino caproic acid is an example of a bifunctional linkergroup. When amino caproic acid is used as part of the linker group, itis not counted as an amino acid in the numbering scheme of theoligopeptide.

The optionally present linker group is not cleavable by TOP, i.e. it isnot cleavable by TOP under physiological conditions.

Prodrug Design

A method of designing a prodrug is another aspect of the invention andentails initially identifying an oligopeptide as described above. Thenthe oligopeptide is linked at a first attachment site of theoligopeptide to a stabilizing group that hinders cleavage of theoligopeptide by enzymes present in whole blood, and directly orindirectly linked to a therapeutic agent at a second attachment site ofthe oligopeptide. The linkage of the oligopeptide to the therapeuticagent and the stabilizing group may be performed in any order orconcurrently. The resulting conjugate is tested for cleavability by TOP.Test compounds resistant to cleavage by TOP are selected. The resultingconjugate may also be tested for stability in whole blood. Testcompounds stable in whole blood are selected.

The first attachment site is usually the N-terminus of the oligopeptidebut may be the C-terminus of the oligopeptide or another part of theoligopeptide. The second attachment site is usually the C-terminus ofthe oligopeptide, but may be the N-terminus of the oligopeptide oranother part of the oligopeptide. A prodrug designed by such a method isalso part of the invention.

Further, the invention includes a method for decreasing toxicity of atherapeutic agent that is intended for administration to a patient.Specifically, a modified, prodrug form of the therapeutic agent isformed by directly or indirectly linking the therapeutic agent to anoligopeptide resistant to cleavage by a trouase, or more specifically,resistant to cleavage by TOP. The oligopeptide is also linked to astabilizing group. The prodrug thus formed provides for decreasedtoxicity of the therapeutic agent when administered to the patient. Themodification of the therapeutic agent in this manner also allows foradministration of an increased dosage of the therapeutic agent to thepatient relative to the dosage of the therapeutic agent in unconjugatedform.

Pharmaceutical Compositions

The invention also includes a pharmaceutical composition comprising acompound, particularly a prodrug compound, according to the inventionand, optionally, a pharmaceutically acceptable carrier, adjuvant,vehicle, or the like.

The invention also relates to the use of the pharmaceutical compositionfor the preparation of a medicinal product intended for the treatment ofa medical condition.

The pharmaceutical composition may, for example, be administered to thepatient parenterally, especially intravenously, intramuscularly, orintraperitoneally. Pharmaceutical compositions of the invention forparenteral administration comprise sterile, aqueous or nonaqueoussolutions, suspensions, or emulsions. As a pharmaceutically acceptablesolvent or vehicle, propylene glycol, polyethylene glycol, injectableorganic esters, for example ethyl oleate, or cyclodextrins may beemployed. Isotonic saline may be part of the pharmaceutical composition.Isotonic saline may be part of the pharmaceutical composition. Thesecompositions can also comprise wetting, emulsifying and/or dispersingagents.

The sterilization may be carried out in several ways, for example usinga bacteriological filter, by incorporating sterilizing agents in thecomposition or by irradiation. They may also be prepared in the form ofsterile solid compositions that may be dissolved at the time of use insterile water or any other sterile injectable medium.

The pharmaceutical composition may also comprise adjuvants which arewell known in the art (e.g., vitamin C, malic acid, antioxidant agents,etc.) and capable of being used in combination with the compound of theinvention in order to improve and prolong the treatment of the medicalcondition for which they are administered.

Doses for administration to a patient of the compounds according to theinvention are generally at least the usual doses of the therapeuticagents known in the field, described in Bruce A. Chabner and Jerry M.Collins, Cancer Chemotherapy, Lippincott Ed., ISBN 0-397-50900-6 (1990)or they may be adjusted, within the judgment of the treating physician,to accommodate the superior effectiveness of the prodrug formulations orthe particular circumstances of the patient being treated. Hence, thedoses administered vary in accordance with the therapeutic agent usedfor the preparation of the compound according to the invention.

Treatment with Prodrug Compound

A method for the therapeutic treatment of a medical condition thatinvolves administering, preferably parenterally and more preferablyintravenously, to the patient a therapeutically effective dose of thepharmaceutical composition is also within the scope of the invention.Thus, a method for treating a patient includes administering to thepatient a therapeutically effective amount of a compound comprising:

(1) a therapeutic agent capable of entering a target cell,

(2) an oligopeptide of the formula (AA)_(n)-AA³-AA²-AA¹, wherein:

each AA independently represents an amino acid,

n is 0 or 1, and when n is 1, then (AA)_(n) is AA⁴ which represents anyamino acid,

AA³ represents isoleucine,

AA² represents any amino acid, and

AA¹ represents any amino acid,

(3) a stabilizing group, and

(4) optionally, a linker group not cleavable by a trouase,

wherein the oligopeptide is directly linked to the stabilizing group ata first attachment site of the oligopeptide and the oligopeptide isdirectly linked to the therapeutic agent or indirectly linked throughthe linker group to the therapeutic agent at a second attachment site ofthe oligopeptide,

wherein the stabilizing group hinders cleavage of the compound byenzymes present in whole blood, and

wherein the compound is cleavable by an enzyme associated with thetarget cell, the enzyme being other than a trouase.

The prodrug compound is useful for the treatment of many medicalconditions including cancer, neoplastic diseases, tumors, inflammatorydiseases, and infectious diseases. Examples of preferred diseases arebreast cancer, colorectal cancer, liver cancer, lung cancer, prostatecancer, ovarian cancer, brain cancer, and pancreatic cancer. Formulatedin pharmaceutically acceptable vehicles (such as isotonic saline), theprodrug compound can be administered to animals or humans in intravenousdoses ranging from 0.05 mg/kg/dose/day to 300 mg/kg/dose/day. It canalso be administered via intravenous drip or other slow infusion method.

Human patients are the usual recipients of the prodrug of the invention,although veterinary usage is also contemplated.

Diagnosis or Assay

An article of manufacture, such as a kit, for diagnosis or assay is alsowithin the scope of the invention. Such an article of manufacture wouldpreferably utilize a compound as described above, except that a marker,such as coumarin is conjugated to the oligopeptide and stabilizing groupinstead of a therapeutic agent. A marker intends any moiety that can beconjugated to the oligopeptide and is readily detectable by any methodknown in the art. At least one reagent useful in the detection of themarker is typically included as part of the kit. Thus, the article ofmanufacture would include the following:

(1) a compound comprising:

(a) a marker,

(b) an oligopeptide of the formula (AA)_(n)-AA³-AA²-AA¹, wherein:

-   -   each AA independently represents an amino acid,    -   n is 0 or 1, and when n is 1, then (AA)_(n) is AA⁴ which        represents any amino acid,    -   AA³ represents isoleucine,    -   AA² represents any amino acid, and    -   AA¹ represents any amino acid,

(c) a stabilizing group, and

(d) optionally, a linker group not cleavable by TOP,

wherein the oligopeptide is directly linked to the stabilizing group ata first attachment site of the oligopeptide and the oligopeptide isdirectly linked to the marker or indirectly linked through the linkergroup to the marker at a second attachment site of the oligopeptide,

wherein the stabilizing group hinders cleavage of the compound byenzymes present in whole blood, and

wherein the compound is cleavable by an enzyme associated with thetarget cell, the enzyme associated with the target cell-being other thanTOP, and

(2) optionally at least one reagent useful in the detection of saidmarker.

The compound is preferably resistant to cleavage by a trouase,especially TOP. The article of manufacture may be used, for example,with patient samples to diagnose tumors or to identify patientssusceptible to treatment by prodrug therapy.

Process Chemistry General Procedures

Oligopeptide: General Method for the Synthesis of Peptides

The peptide, or oligopeptide, sequences in the prodrug conjugates ofthis invention may be synthesized by the solid phase peptide synthesis(using either Boc or Fmoc chemistry) methods or by solution phasesynthesis. The general Boc and Fmoc methods are widely used and aredescribed in the following references: Merrifield, J. A. Chem. Soc.,88:2149 (1963); Bodanszky and Bodanszky, The Practice of PeptideSynthesis, Springer-Verlag. Berlin, 7-161 (1994); Stewart, Solid PhasePeptide Synthesis, Pierce Chemical, Rockford, (1984).

General Fmoc Solid Phase Method

Using the preferred solid phase synthesis method, either automated ormanual, a peptide of desired length and sequence is synthesized throughthe stepwise addition of amino acids to a growing chain which is linkedto a solid resin. Examples of useful Fmoc compatible resins include, butare not limited to, Wang resin, HMPA-PEGA resin, Rink acid resin, or ahydroxyethyl-photolinker resin. The C-terminus of the peptide chain iscovalently linked to a polymeric resin and protected α-amino acids wereadded in a stepwise manner with a coupling reagent. A preferred α-aminoprotecting group is the Fmoc group, which is stable to couplingconditions and can readily be removed under mild alkaline conditions.The reaction solvents are preferably but not limited to DMF, NMP, DCM,MeOH, and EtOH. Examples of coupling agents are: DCC, DIC, HATU, andHBTU. Cleavage of the N-terminal protecting group is accomplished in10-100% piperidine in DMF at 0-40° C., with ambient temperature beingpreferred. At the end of synthesis the final Fmoc protecting group isremoved using the above N-terminal cleavage procedure. The remainingpeptide on resin is cleaved from the resin along with any acid sensitiveside chain protecting groups by treating the resin under acidicconditions. For example, an acidic cleavage condition is a mixture oftrifluoroacetic acid (TFA) in dichloromethane. If thehydroxyethyl-photolinker resin is used, the appropriate wavelength forinducing cleavage is λ365 nm ultraviolet light. A diagrammaticrepresentation of this process is given in FIG. 3.

General N-Cap Method Via Solid Phase Synthesis

The preparation of N-terminus derivatized peptides is convenientlyaccomplished on solid phase. When the peptide synthesis is complete, theterminal Fmoc is removed while the peptide is still on the solidsupport. The N-cap of choice is coupled next using standard peptidecoupling conditions onto the N-terminus of the peptide. On completion ofthe N-cap coupling the peptide is cleaved from the resin using theprocedure described above if the Fmoc synthesis procedure is used.

General Boc Solid Phase Method

For the solid phase method using Boc chemistry, either the Merrifieldresin or PAM resin is useful. The amino acids are coupled to the growingchain on solid phase by successive additions of coupling agent activatedBoc-protected amino acids.

Examples of coupling agents are: DCC, DIC, HATU, and HBTU. The reactionsolvents may be DMF, DCM, MeOH, or NMP. Cleavage of the Boc protectinggroup is accomplished in 10-100% TFA in DCM at 0-40° C., with ambienttemperature being preferred. On completion of the peptide chain assemblythe N-terminus protecting group (usually Boc) is removed as describedabove. The peptide is removed from the resin using liquid HF ortrifluoromethane sulfonic acid in dichloromethane.

General Procedure for the Preparation of Fmoc Oligopeptide by SolutionPhase Synthesis

Alternatively, the prodrug peptide intermediate may be made via asolution phase synthesis, utilizing either Boc or Fmoc chemistry. In thediagrammatic presentation of the methods (FIG. 4), the C-terminal Leutetrapeptide is generally used as an example, but it will be understoodthat similar reactions may be performed with other C-terminaltetrapeptides, as well. The peptide can be built up by the stepwiseassembly in analogy to the solid phase method (in the N-terminaldirection or in the C-terminal direction) or through the coupling of twosuitably protected dipeptides or a tripeptide with a single amino acid.

One method of solution phase synthesis is a stepwise building up of theprodrug peptide intermediate using Fmoc chemistry, shown in FIG. 4. TheC-terminus must be protected to reduce the formation of side products.The C-terminal R group in FIG. 4 is Me, tBu, benzyl or TCE. (Note thatwhen the N-cap is methyl succinyl, the C-terminus R group cannot beMethyl.) Although DMF is given as the solvent, other solvents such asDMSO, CH₃CN, or NMP (or mixtures thereof) may be substituted therefor.Pyridine, Et₃N or other bases may be substituted for piperidine indeprotecting the growing peptide chain protected amino terminus.Similarly, although HBTU is given in the diagram above as the activatingagent, other activating agents such as DCC, DIC, DCC+HOBt, OSu,activated esters, azide, or triphenyl phosphoryl azide may be used.Additionally, the protected peptide acid chloride or acid bromide may beused to couple directly to the amino acid or peptide fragment. Oncompletion of the oligopeptide assembly, the N-terminus is deprotectedand the C-terminus protected peptide is ready to accept the desiredN-cap.

General Procedure for the Preparation of N-Capped Oligopeptide viaSolution Phase Synthesis

When constructing the N-capped oligopeptide by solution phase synthesis,the N-cap needs to be synthesized by a slightly modified procedure (FIG.4). First the C-terminus of the Fmoc oligopeptide needs to be protectedwith an acid labile or hydrogenation sensitive protecting groupcompatible with the selective deprotection of the C-terminus over theN-cap. Then the Fmoc protecting group needs to be removed from theoligopeptide to reveal the N-terminus. With the N-terminus deprotectedand the C-terminus protected, the oligopeptide is reacted with theactivated hemiester of the desired N-cap. The N-cap can be activatedusing methods for activating amino acids such as DCC or HATU in base andan appropriate solvent. Alternatively, where the methyl-hemisuccinate isused, the coupling may also be done via methyl hemisuccinyl chloride (orother acid halide) (FIG. 4) using an inert solvent in the presence of anorganic or inorganic base, such as DIEA, triethylamine or Cs₂CO₃. Oneexample of such a synthesis includes reacting methyl-hemisuccinate andβAla-Ile-Ala-Leu benzyl ester. The coupling method can be any one of themethods generally used in the art (see for example: Bodanszky, M., ThePractice of Peptide Synthesis, Springer Verlag, 185 (1984); Bodanszky,M., Principles of Peptide Synthesis, Springer Verlag, 159 (1984). Thebenzyl group then can be removed by catalytic hydrogenation providingthe desired N-cap methyl-succinyl form of oligopeptide no. 8. Otherexamples of suitable, selectively removable C-terminal protecting groupscan be, but are not limited to, tBu, alkoxy-methyl and TCE. Othermethods of accomplishing this step are described in the literature.

Any combination of the above method can be considered, such as “fragmentcondensation” of di-, or tripeptides. The reaction conditions are wellknown in the art and detailed in the citations given. The advantage ofthe above described methods is the facile purification of the productproduced by solution phase synthesis.

Prodrug Conjugate

General Methods for the Conjugation and Deprotection Steps

The N-cap form of the oligopeptide therapeutic agent described in thisinvention can be synthesized by coupling an Fmoc form (which means Fmocis attached to the N-terminus of the oligopeptide) of the oligopeptidewith daunorubicin, doxorubicin, or any appropriate therapeutic agentusing any of the standard activating reagents used in peptide synthesis(FIG. 5). The solvent may be toluene, ethyl acetate, DMF, DMSO, CH₃CN,NMP, THF, DCM or any other suitable inert solvent as is known in the artand the reagents are soluble therein. The preferred solvents are DMF andNMP. The appropriate temperature range is −25 to +25° C., with ambienttemperature being preferred. The activating agent may be selected fromone of the following: PyBOP, HBTU, HATU, EDC, DIC, DCC, DCC+HOBT, OSuactivated esters, azide, or triphenylphosphorylazide. HBTU or HATU isthe preferred activating agent. Alternatively, the acid chloride or theacid bromide of the protected peptide can also be used for this couplingreaction. 2-4 equivalent, advantageously 2-2.5 equivalent of a base isrequired for the coupling reaction. The base can be selected frominorganic bases such as CsCO₃, Na₂CO₃, or K₂CO₃, or organic bases, suchas TEA, DIEA, DBU, DBN, DBO, pyridine, substituted pyridines,N-methyl-morpholine etc., preferably TEA, or DIEA. The reaction can becarried out at temperatures between −15° C. and 50° C., advantageouslybetween −10° C. and 10° C. The reaction time is between 5-90 minutes andis advantageously 20-40 minutes. The product is isolated by pouring thereaction mixture into water and filtering the precipitate formed. Thecrude product can be further purified by recrystallization from DCM,THF, ethyl acetate, or acetonitrile, preferably from dichloromethane oracetonitrile. The isolated Pmoc form of the oligopeptide therapeuticagent conjugate is then deprotected over 2-90 minutes, preferably 3-8minutes, using a ten- to hundred-fold excess of base at a temperaturebetween −10° C. and 50° C. Ideally, 5-60 equivalents of the base arepreferred. Piperidine is the preferred base to deprotect Fmoc groups.The deprotected amino terminus of the oligopeptide-therapeutic agentconjugate is acylated by a diacid anhydride or a hemi protectedactivated diacid (i.e., a mono ester which is subsequently deprotected)to give the final N-cap form of the oligopeptide-therapeutic agent.

Alternatively, the final prodrug can be similarly prepared from theprotected N-cap form of the oligopeptide such as a methyl hemiester formof succinyl-N-cap oligopeptide and conjugated to a therapeutic agent.This method is illustrated in FIG. 6.

The protected N-Cap-oligopeptide-therapeutic agent is now deprotected bymethods compatible with the stability of the therapeutic agent. Forexample, anthracyclines may be protected with a methyl group anddeprotected with an esterase. For other therapeutic agents, benzylprotecting groups and catalytic hydrogenation may be selected fordeprotection.

The salt form of the negatively charged N-cap oligopeptide-therapeuticagent is carried out with a solvent selected from the following group:alcohol (including methanol, ethanol, or isopropanol), water,acetonitrile, tetrahydrofuran, diglyme or other polar solvents. Thesodium source is one molar equivalent of NaHCO₃, NaOH, Na₂CO₃, NaOAc,NaOCH₃ (in general sodium alkoxide), or NaH. An ion exchange columncharged with Na⁺ (such as strong or weak ion exchangers) is also usefulfor this last step of making the salt form of the N-cap oligopeptidetherapeutic agent when appropriate. Sodium is described as an exampleonly.

Generally, the prodrug may be converted to a pharmaceutically acceptablesalt form to improve solubility of the prodrug. The N-cap-oligopeptidetherapeutic agent is neutralized with a pharmaceutically acceptablesalt, e.g., NaHCO₃, Na₂CO₃, NaOH tris(hydroxymethyl)aminomethane, KHCO₃,K₂CO₃, CaCO₃, NH₄OH, CH₃NH₂, (CH₃)₂NH, (CH₃)₃N, acetyltriethylammonium.The preferred salt form of prodrug is sodium, and the preferredneutralizing salt is NaHCO₃.

It is well documented that anthracycline type molecules, includingdoxorubicin and daunorubicin form gels in organic solvents in very lowconcentrations (Matzanke, B. F., et al., Eur. J. Biochem., 207:747-55(1992); Chaires, J. B., et al., Biochemistry, 21:3927-32 (1982);Hayakawa, E., et al., Chem. Pharm. Bull., 39:1282-6 (1991). This may bea considerable obstacle to getting high yields of clean product whenmaking peptide anthracycline conjugates. The gel formation contributesto the formation of undesirable side reactions. One way to minimize thisproblem is to use very dilute solutions (1-2%) for the couplingreaction, however it is not practical in a process environment (largeamounts of waste, complicated isolation). To overcome this problem ureaor other chaotropic agents may be used to break up the stronghydrophobic and hydrogen bonding forces forming the gel. Thus if thecoupling reaction is carried out in a urea-containing solvent,advantageously a 20% to saturated solution of urea in DMF or NMP, theside reactions can be kept below 2% even if the concentration ofreactants exceeds 10%. This makes the conjugation step practical at highconcentrations and produces good yields.

General Enzyme Method

Hydrolysis of protected N-cap-oligopeptide therapeutic agents to thefull N-cap compound catalyzed by acids or bases leads to complexreaction mixtures due to the ability of many therapeutic agents evenunder moderately acidic or basic conditions. Enzymes can promote thehydrolysis without destroying the substrate or the product. Enzymessuitable for this reaction can be esterases or lipases and can be intheir natural, water soluble forms or immobilized by cross coupling, orattachment to commercially available solid support materials. Of thesoluble enzymes evaluated, Candida Antarctica “B” lipase (AltusBiologics) is especially useful. An example of an enzyme immobilized bycross coupling is ChiroCLEC-PC™ (Altus Biologics). Candida Antarctica“B” lipase (Altus Biologics) can be immobilized by reaction with NHSactivated Sepharose™ 4 Fast Flow (American Pharmacia Biotech). The pH ofthe reaction mixture during the hydrolysis is carefully controlled andmaintained by a pH-stat between 5.5 and 7.5, advantageously between 5.7and 6.5, via controlled addition of NaHCO₃ solution. When the reactionis completed the product is isolated by lyophilization of the filteredreaction mixture. The immobilized enzymes remain on the filter cake andcan be reused if desired.

General Allyl or Alkyl Ester Method

The prodrug can also be prepared via coupling an allyl-hemiester oralkyl hemiester form of the N-cap oligopeptide with a therapeutic agentand then liberating the free acid from the conjugate. FIG. 8 illustratesthis process with Succinyl-β-Ala-Ile-Ala-Leu (SEQ ID NO:44) anddoxorubicin.

The coupling of allyl-succinyl-βAla-Ile-Ala-Leu (SEQ ID NO:45) withdoxorubicin can be carried out via any one of the oligopeptideconjugation methods. Allyl-succinyl-βAla-Ile-Ala-Leu-doxorubicin (SEQ IDNO:46) can also be synthesized by reacting allyl hemisuccinate, whichwas prepared via known methods (Casimir, J. R., et al., Tet. Lett. 36/193409 (1995)), with βAla-Ile-Ala-Leu-doxorubicin (SEQ ID NO:40) similarlyas coupling of the protected tetrapeptide precursors to doxorubicin wasdescribed in the previous methods, shown in FIG. 5. Suitable inertsolvents are THF, dichloromethane, ethyl acetate, toluene, preferablyTHF from which the acid form of the product precipitates as the reactionprogresses. The isolated acid is converted to its sodium salt asdescribed earlier. Reaction times vary between 10-180 minutes,advantageously 10-60 minutes, at temperatures between 0-60° C.,preferably 15-30° C.

Removal of the allyl or alkyl group can be done with Pd(0), or Ni(0),advantageously Pd(0) promoted transfer of the allyl or alkyl group toacceptor molecules, as it is well known in the art and documented in thescientific literature (Genet, J-P, et al., Tet. Lett., 50, 497, 1994;Bricout, H., et al. Tet. Lett., 54:1073 (1998), Genet, J-P. et al.Synlett, 680 (1993); Waldmann, H., et al., Bioorg. Med. Chem., 7:749(1998); Shaphiro, G., Buechler, D., Tet. Lett., 35:5421 (1994)). Theamount of catalyst can be 0.5-25 mol % to the substrate.

General Trityl or Substituted Trityl Method

The prodrug may also be synthesized via the method shown in FIG. 7. Thisapproach utilizes an R′-oligopeptide, where R′ is trityl or substitutedtrityl. The coupling of R′-oligopeptide with a therapeutic agent can becarried out via any one of the methods described earlier for conjugationof a protected oligopeptide with a therapeutic agent at 30-120 minutesat 0-20° C.

Removal of trityl or substituted trityl group can be achieved underacidic conditions to give the positively charged prodrug. Thispositively charged prodrug is N-capped as illustrated in FIG. 4 anddescribed earlier. The trityl deprotection can be accomplished withacetic acid, formic acid and dilute hydrochloric acid.

The prodrug can be converted into (succinyl orglutaryl)-oligopeptide-therapeutic agent by reacting with succinicanhydride or glutaric anhydride, then further converted into anypharmaceutically acceptable salt. The solvent for the coupling step maybe DMF, DMSO, CH₃CN, NMP, or any other suitable solvent is known in theart.

General Inverse Direction Solid Phase Conjugation Method

The prodrug compound of the present invention can be synthesized byusing solid phase chemistry via “step wise” inverse (from the N-terminalto the C-terminal) direction methods.

One way is to use resins to immobilize a succinyl hemiester, for examplesuccinyl-mono-benzyl ester or -allyl ester. Examples of resins could beselected are “Wang Resins” (Wang, S. S., J. Am. Chem. Soc., 95:1328(1973); Zhang, C., Mjaili, A. M. M., Tet. Lett., 37:5457 (1996)), “RinkResins” (Rink, H., Tet. Lett., 28:3787 (1987)), “Trityl-, orsubstituted-trityl Resins” (Chen, C., et al., J. Am. Chem. Soc.,116:2661 (1994); Bartos, K. et al., Peptides, Proc. 22^(nd) EuropeanPeptide Symposium (1992); Schneider, C. H.; Eberle, A. N. (Eds.), ESCOM,Leiden, pp. 281 (1993). The immobilized ester is then deprotected andreacted with, for example, a similarly C-terminal protected β-alanine.These steps are then repeated with isoleucine, alanine, and finallyleucine esters, followed by the coupling of doxorubicin to theimmobilized succinyl-tetrapeptide. The molecule is then liberated fromthe resin by using mildly acidic conditions to form a free prodrug, suchas Suc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:39). This methodology isrepresented on the scheme of FIG. 9. Another version of phase synthesisutilizes immobilized succinyl oligopeptide ester. This is thenC-terminally deprotected, followed by the coupling step to doxorubicinor other therapeutic agent and finally liberated from the resin asrepresented on the scheme of FIG. 9. The acid form of the prodrugmolecules may then be converted finally into its sodium salt asdescribed above.

Removal of Free Therapeutic Agent

Unconjugated therapeutic agent may be present late in the process ofmaking the prodrug. For example, during the coupling step of(stabilizing group)-(oligopeptide) conjugate with doxorubicin as thetherapeutic agent, it was found, in some instances, that the reactiondid not proceed completely. There was about 2-4% of residual doxorubicinremaining in the coupled product. Initial attempts to remove doxorubicincompletely from the product by acidic washes did not result in completeremoval. The complete removal of the free therapeutic agent was effectedby the process outlined in Example 21 and FIG. 17 that utilizesscavenging resin or beads.

The crude product, which contains the intermediate and residualdoxorubicin, were dissolved in DMF and polystyrene methylisocyanate orpolystyrene sulfonyl chloride resin or beads were added. The reactionwas stirred for 60 minutes. The free amino group of doxorubicin reactswith the isocyanate or sulfonyl chloride group on the beads to form aurea or sulfonamide derivative. The solid beads with doxorubicinattached to them were then separated from the desired product byfiltration. The desired product remains in the DMF solution. Thisapproach seems to be a very mild and effective method for removingresidual therapeutic agent from the product.

General Large Scale Compound Synthesis

The prodrug compound can be synthesized using a simple and efficientthree-step process of the invention: (1) coupling an alkyl or allylester protected stabilizing group-oligopeptide and a therapeutic agentin the presence of an activating agent to make an alkyl or allyl esterprotected stabilizing group-oligopeptide-therapeutic agent conjugate,(2) removing uncoupled therapeutic agent that remains after the couplingstep, and (3) deprotecting the alkyl or allyl ester protectedstabilizing group-oligopeptide-therapeutic agent conjugate to make thestabilizing group-oligopeptide-therapeutic agent prodrug compound.

The first step involves the coupling of an alkyl-ester protectedoligopeptide fragment to a therapeutic agent. A preferred embodiment ofthe first step involves the coupling of an alkyl or allyl esterprotected stabilizing group oligopeptide, such asMeOSuc-βAla-Ile-Ala-Leu-OH (SEQ ID NO:47), with a therapeutic agent,such as doxorubicin, using an activating agent, such as HATU, to givealkyl or allyl ester protected stabilizing group oligopeptidetherapeutic agent conjugate, e.g., MeOSuc-βAla-Ile-Ala-Leu-Dox (SEQ IDNO:48). The focus of this step is on the purity and the yield of themethyl ester, since it was found that the hydrolysis step did not havean impact on purity. Preferably the molar ratio of the alkyl or allylester protected stabilizing group oligopeptide to the therapeutic agentwill be between 2:1 and 1:1. More preferably the molar ratio is between1.75:1 and 1.5:1. Most preferably the molar ratio is 1.66:1.

The coupling of the alkyl or allyl ester protected stabilizing groupoligopeptide and a therapeutic agent is preferably performed by: (a)combining the alkyl or allyl ester protected stabilizing groupoligopeptide and the therapeutic agent in DMF, (b) adding DIEA, (c)reacting the alkyl or allyl ester protected stabilizing groupoligopeptide and the therapeutic agent in the presence of the activatingagent to form the conjugate, and (d) precipitating the conjugate byadding a brine solution to form a precipitate. Preferably the molarratio of the DIEA and the alkyl or allyl ester protected stabilizinggroup-oligopeptide is between 3:1 and 1.5:1. More preferably the molarratio is 2.5:1 and 2:1. Most preferably the molar ratio is 2.18:1. Thereacting step is preferably performed at 0° C., for 30 minutes.Preferably the molar ratio of the activating agent and the alkyl orallyl ester protected stabilizing group-oligopeptide is between 1.5:1and 1:1. More preferably, the molar ratio is 1.1:1. The brine solutionis preferably between 20% (w/v) and 40% (w/v) of NaCl in water. Morepreferably the brine solution is preferably between 25% (w/v) and 35%(w/v) of NaCl in water. Most preferably the brine solution is 30% (w/v)of NaCl in water. The conjugate is preferably precipitated in a brinesolution, wherein the pH is between 5.0 and 7.0, inclusive. Mostpreferably, the conjugate is precipitated at a pH between 5.8 and 6.0.

Since many therapeutic agents are toxic substances, it is preferable toeliminate any free therapeutic agent from the coupled product. Theremoving step is preferably performed by: (a) dissolving the conjugatein DMF, (b) dissolving a scavenger resin in anhydrous DMF, (c) addingthe alkyl or allyl ester protected stabilizing group oligopeptidetherapeutic agent conjugate formed in the coupling step to the scavengerresin to form a conjugate-resin mixture, (d) maintaining the mixture atbetween 0° C. and 30° C. for 2 to 24 hours wherein the uncoupledtherapeutic agent reacts with the resin, (e) removing the resin from themixture, and (f) precipitating the remainder by adding a brine solutionto form a precipitate of the alkyl or allyl ester protected stabilizinggroup oligopeptide therapeutic agent conjugate. Preferably the scavengerresin is polystyrene-isocyanate (PS-isocyanate), PS-methylisocyanate,PS-thioisocyanate, PS-methylthioisocyanate, PS-sulfonyl chloride,PS-methylsulfonyl chloride or PS-benzaldehyde. Most preferably, thescavenger resin is PS-isocyanate. The removing step is preferablyperformed to remove free therapeutic agent, which is an anthracycline.

The third step is deprotecting the alkyl or allyl ester protectedstabilizing group-oligopeptide-therapeutic agent conjugate, preferablyvia hydrolysis by an enzyme, more preferably via hydrolysis by anesterase, which directly gives the prodrug compound in good yield with afinal purity of at least 90%. For example, the third step may be thehydrolysis of the methyl ester group in MeOSuc-βAla-Ile-Ala-Leu-Dox (SEQID NO:48) by an enzyme, such as CLEC CAB (crosslinked Candida AntarticaB Lipase), which directly gives the sodium salt ofSuc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:39) in quantitative yields with highpurity.

The enzyme is preferably either crosslinked or immobilized on a solidsupport. The esterase may be pig liver esterase, Candida Antartica BLipase, Candida Rugosa lipase, Pseudomonas Cepacia lipase, pig liveresterase immobilized on sepharose, Candida antartica B lipaseimmobilized on sepharose, CLEC-PC™ (Pseudomonas Cepacia lipase),CLEC-CAB (Candida Antartica B lipase), or CLEC-CR (Candida Rugosalipase). Deprotecting via hydrolysis by an enzyme is preferablyperformed by: (a) washing the enzyme to remove free enzyme, (b) addingthe washed enzyme to the alkyl or allyl ester protected stabilizinggroup-oligopeptide-therapeutic agent conjugate, (c) reacting the enzymewith the conjugate at between 15° C. and 40° C., inclusive, at a pHbetween 5.0 and 8.0, inclusive, for at least 18 hours, to create thestabilizing group-oligopeptide-therapeutic agent prodrug compound, and(d) separating the enzyme from the prodrug compound. Most preferablyadditional washed crosslinked or immobilized enzyme is added after thestep of reacting the enzyme with the conjugate, prior to separating theenzyme from the prodrug compound.

Thus, the invention includes a method of making a compound comprising:

(1) selecting an Fmoc-protected oligopeptide of the formulaFmoc-(AA)_(n)-AA³-AA²-AA¹, wherein:

each AA independently represents an amino acid,

n is 0 or 1, and when n is 1, then (AA)_(n) is AA⁴ which represents anyamino acid,

AA³ represents isoleucine,

AA² represents any amino acid, and

AA¹ represents any amino acid,

(2) coupling the Fmoc-protected oligopeptide to a therapeutic agent byactivating the Fmoc-protected oligopeptide with an activating agent inthe presence of the therapeutic agent to form an Fmoc-protectedoligopeptide-therapeutic agent conjugate,

(3) deprotecting the Fmoc-protected oligopeptide-therapeutic agentconjugate by contacting it with a base to form anoligopeptide-therapeutic agent conjugate, and

(4) coupling the oligopeptide-therapeutic agent conjugate to astabilizing group to form the compound.

Alternatively, a method of making a compound comprises the followingsteps:

(1) selecting an oligopeptide having a formula (AA)_(n)-AA³-AA²-AA¹wherein:

each AA independently represents an amino acid,

n is 0 or 1, and when n is 1, then (AA)_(n) is AA⁴ which represents anyamino acid,

AA³ represents isoleucine,

AA² represents any amino acid, and

AA¹ represents any amino acid,

(2) coupling the oligopeptide to an alkyl ester-protected stabilizinggroup to form an alkyl ester-protected stabilizing group-oligopeptideconjugate,

(3) coupling the alkyl ester-protected-stabilizing group-oligopeptideconjugate to a therapeutic agent by activating the alkyl ester-protectedstabilizing group-oligopeptide conjugate with an activating agent in thepresence of a therapeutic agent to form an alkyl ester-protectedstabilizing group-oligopeptide-therapeutic agent conjugate, and(4) deprotecting the alkyl ester-protected stabilizinggroup-oligopeptide therapeutic agent conjugate to form the compound.

A compound of the invention may also be made via the following steps:

(1) selecting an oligopeptide of the formula (AA)_(n)-AA³-AA²-AA¹,wherein:

each AA independently represents an amino acid,

n is 0 or 1, and when n is 1, then (AA)_(n) is AA⁴ which represents anyamino acid,

AA³ represents isoleucine,

AA² represents any amino acid, and

AA¹ represents any amino acid,

(2) coupling the oligopeptide to an allyl ester-protected stabilizinggroup to form an allyl ester-protected stabilizing group-oligopeptideconjugate,

(3) coupling the allyl ester-protected-stabilizing group-oligopeptideconjugate to a therapeutic agent by activating the allyl ester-protectedstabilizing group-oligopeptide conjugate with an activating agent in thepresence of a therapeutic agent to form an allyl ester-protectedstabilizing group-oligopeptide-therapeutic agent conjugate, and(4) deprotecting the allyl ester-protected stabilizinggroup-oligopeptide therapeutic agent conjugate to form the compound.

Yet another method for making a compound of the invention comprises thefollowing steps:

(1) selecting a trityl-protected oligopeptide of the formulatrityl-(AA)_(n)-AA³-AA²-AA¹, wherein:

each AA independently represents an amino acid,

n is 0 or 1, and when n is 1, then (AA)_(n) is AA⁴ which represents anyamino acid,

AA³ represents isoleucine,

AA² represents any amino acid, and

AA¹ represents any amino acid,

(2) coupling the trityl-protected oligopeptide to a therapeutic agent byactivating the trityl-protected oligopeptide with an activating agent inthe presence of a therapeutic agent, thereby making a trityl-protectedoligopeptide-therapeutic agent conjugate,(3) deprotecting the trityl-protected oligopeptide-therapeutic agentconjugate under acidic conditions to form an oligopeptide-therapeuticagent conjugate, and(4) coupling the oligopeptide-therapeutic agent conjugate with anstabilizing group to form the compound.

Another possible step in connection with any of these methods isremoving uncoupled therapeutic agent by use of scavenging resin orbeads. Further, the compound may be neutralized with a pharmaceuticallyacceptable salt if desired.

Specific Compounds

Compounds of the invention include the prodrugs specifically listed inTable 1 (under Example 1).

EXAMPLES Example 1 Screening of Potential Prodrugs

A good candidate for a prodrug with improved therapeutic index isactivated by cancer cells but relatively stable in whole human blood.Three different preparations of carcinoma cell trouase were used toscreen various test compounds. These three preparations were as follows:

(a) MCF 7/6 (breast carcinoma) cell homogenate

(b) MCF 7/6 (breast carcinoma) conditioned media, and

(c) HeLa (cervical carcinoma) cell extract anion exchange fraction pool.

a. Preparation of MCF 7/6 Cell Homogenate

MCF 7/6 cells were grown to confluence in a serum free medium containingDMEM:F12 (1:1), 50 mg/L bovine serum albumin, ITS-X (10 mg/L insulin,5.5 mg/L transferrin, 6.7 μg/L Na selenite, 2 mg/L ethanolamine), andLipid Concentrate (Gibco #21900-030)100 mL of cells were harvested bycentrifugation at 4° C. 10,000×g, for 20 min and decanting thesupernatant. The pellet was resuspended in 2 mL phosphate bufferedsaline (Gibco) and centrifuged at 18,000×g for 10 min. After decantingthe supernatant, the cells (approximately 300 μL wet) were homogenizedby grinding in 1.7 mL 10 mM pH 7.2 HEPES buffer (sodium salt). Thehomogenate was centrifuged at 18,000×g at 4° C. for 5 min and thesupernatant was aliquoted and stored at ≦−20° C. for subsequent use inthe compound screen.

b. Preparation of MCF 7/6 Conditioned Media

MCF 7/6 cells were grown to confluence in DMEM/F12 (1:1) mediumcontaining 10% fetal bovine serum, 0.05% (w/v) L-glutamine, 250 IU/mLpenicillin, and 100 μg/mL streptomycin. Cells were then washed twicewith phosphate buffered saline and incubated 24 hr at 5% CO₂, 37° C., inDMEM/F12 (1:1), 0.02% BSA, ITS-X (10 mg/L insulin, 5.5 mg/L transferrin,6.7 μg/L Na selenite, 2 mg/L ethanolamine). The conditioned media wasthen decanted and, using a stirred cell apparatus with a YM10 (10,000 MWcutoff) ultrafiltration membrane (Millipore), exchanged once with 10 mMHEPES buffer, pH 7.2 and concentrated twenty-fold. This solution wasstored in aliquots at −20° C. for use in the compound screen.

c. Preparation of HeLa Cell Anion Exchange Fraction Pool

Thirty billion commercially produced HeLa Cells (human cervicalcarcinoma, Computer Cell Culture Center, Seneffe, Belgium) werehomogenized with a sonicator and with a Dounce homogenizer in 108 mL ofaqueous lysis solution. The lysis solution contained 0.02% w/v TritonX-100, 0.04% w/v sodium azide, and a cocktail of protease inhibitors (2tablets/50 mL Complete™, EDTA-free tablets, Roche MolecularBiochemicals). The cell homogenate was centrifuged 30 minutes at 4° C.at 5000×g and the pellet was homogenized in a second 108 mL of lysissolution using a Dounce homogenizer and centrifuged as before. Thesupernatants were combined and centrifuged for 90 min at 145,000×g at 4°C.

A portion of the ultracentrifugation supernatant was diluted 2-fold witha 20 mM triethanolamine-HCl pH 7.2 buffer containing 0.01% (w/v) TritonX-100 and 0.02% (w/v) sodium azide (equilibration buffer). Thirty mL ofthe resulting solution, corresponding to approximately 180 mg ofprotein, was loaded at 4° C. on a 2.6×9.4 cm Source™15Q (AmershamPharmacia Biotech) low pressure anion exchange chromatography column (1ml/minute). The column was then washed with 250 ml of the equilibrationbuffer at a flow rate of 1 mL/minute. Proteins were eluted in a NaCllinear concentration gradient (0-0.5 M in the equilibration buffer,total volume of the gradient was 1000 ml) at a flow rate of 3 ml/minute.Two-minute fractions were collected and used for enzyme activitydetermination using βAla-Leu-Ala-Leu-Dox (SEQ ID NO:79) as thesubstrate. Its transformation into Ala-Leu-Dox was quantified by reversephase high performance liquid chromatography utilizing fluorescencedetection of the anthracycline moiety. The fractions containing thehighest activity levels were pooled (fractions #43-46; ˜0.13 M NaCl),supplemented with protease inhibitors (Complete™, EDTA-free tablets,Roche Molecular Biochemicals), and stored as aliquots at −80° C.

d. Cleavage Assay

Test compounds were incubated for 2 hr at 37° C. at a concentration of12.5 μg/mL with each of the three different preparations of carcinomacell enzyme. Following incubation, three volumes of acetonitrile wereadded to stop the reaction and remove protein from the mixture. Thesample was centrifuged at 18,000 g for 5 minutes and 100 μL ofsupernatant was mixed with 300 μL of water prior to analysis by HPLC.For HPLC analysis, 50 μL of sample was injected on a 4.6×50 mm 2μ TSKSuper-ODS chromatography column at 40° C. and eluted with a 3 minutelinear gradient from 26% to 68% acetonitrile in aqueous 20 mM ammoniumformate pH 4.5 buffer at 2 mL/min. Detection was by fluorescence usingan excitation wavelength of 235 nm and an emission wavelength of 560 nm.

Test compounds that were not cleaved (less than or equal to 5% ofcontrol) by the enzyme preparations under the given conditions are shownin Table 1 below. With few exceptions, results for carcinoma cell enzymecleavage were identical for a partially purified fraction from HeLacells, MFC 7/6 cell homogenate, and MCF 7/6 conditioned media.

TABLE 1 Stabilizing Therapeutic No: Group (AA₄) (AA₃) (AA₂) (AA₁)Compound 1 Suc βAla Ile Ala Phe Dnr (SEQ ID NO: 49) 2 Suc βAla Ile AlaIle Dnr (SEQ ID NO: 50) 3 Suc Tic Ile Ala Leu Dnr (SEQ ID NO: 51) 4 SucThi Ile Ala Leu Dnr (SEQ ID NO: 52) 5 Suc Nal Ile Ala Leu Dnr (SEQ IDNO: 53) 6 Suc βAla Ile Ala Leu Dnr (SEQ ID NO: 43) 7 Suc Amb Ile Ala LeuDnr (SEQ ID NO: 54) 8 Suc Aib Ile Ala Leu Dnr (SEQ ID NO: 55) 9 Suc βAlaIle Ala Leu Dox (SEQ ID NO: 39) 10 Suc Thi Ile Aib Leu Dnr (SEQ ID NO:56) 11 Suc Nal Ile Aib Leu Dnr (SEQ ID NO: 57) 12 Suc βAla Ile Aib LeuDnr (SEQ ID NO: 58) 13 Suc Amb Ile Aib Leu Dox (SEQ ID NO: 59) 14 SucAib Ile Aib Leu Dnr (SEQ ID NO: 60) 15 Suc βAla Ile Gly Phe Dnr (SEQ IDNO: 61) 16 Suc βAla Ile Gly Ile Dnr (SEQ ID NO: 62) 17 Suc Tic Ile GlyLeu Dnr (SEQ ID NO: 63) 18 Suc Thi Ile Gly Leu Dnr (SEQ ID NO: 64) 19Suc Nal Ile Gly Leu Dnr (SEQ ID NO: 65) 20 Suc βAla Ile Gly Leu Dnr (SEQID NO: 66) 21 Suc Amb Ile Gly Leu Dnr (SEQ ID NO: 67) 22 Suc Aib Ile GlyLeu Dnr (SEQ ID NO: 68) 23 Suc βAla Ile Thr Ile Dnr (SEQ ID NO: 69) 24Suc βAla Ile Tyr Ile Dnr (SEQ ID NO: 70) 25 Suc βAla Ile Tyr Leu Dnr(SEQ ID NO: 71) 26 Suc βAla Ile Tyr Gly Dox (SEQ ID NO: 72) 27 Suc βAlaIle Ala Gly Dox (SEQ ID NO: 73) 28 Suc Ø Ile Ala Leu Dox 29 Suc Ø IleN(Me)Ala Leu Dox 30 Suc Ø Ile Ala Gly Dox Ø = not present

Example 2 Tumor-Activated Prodrug Activity on LNCaP, HT-29 and PC-3Cells

Adherent cells, LNCaP (prostate carcinoma), HT-29 (colon carcinoma) andPC-3 (prostate carcinoma), were cultured in DMEM media containing 10%heat inactivated fetal calf serum (FCS). On the day of the study thecells were detached from the plate with a trypsin solution. Thecollected cells were washed and resuspended at a concentration of0.25×10⁶ cells/ml in DMEM containing 10% FCS. 100 μl of cell suspensionwere added to 96 well plates and the plates were incubated for 3 hoursto allow the cells to adhere. Following this incubation, serialdilutions (3-fold increments) of doxorubicin or test compounds were madeand 100 μl of compounds were added per well. The plates were thenincubated for 24 hours, pulsed with 10 μl of a 100 μCi/ml ³H-thymidineand incubated for an additional 24 hours (total incubation time 48hours). The plates were harvested using a 96 well Harvester (PackardInstruments) and counted on a Packard Top Count Counter. Four parameterlogistic curves were fitted to the ³H-thymidine incorporation as afunction of drug molarity using Prism software to determine IC₅₀ values.

IC50 (μM) Compound LNCaP HT29 PC-3 DOX 0.016 0.052 0.075Suc-Ile-Ala-Leu-Dox 1.1 47 88 Suc-Ile-NMeAla-Leu-Dox 0.51 36 66Suc-Ile-Pro-Leu-Dox 2.0 44 106 Suc-βAla-Ile-Ala-Leu-Dox 0.19 38 57 (SEQID NO:39)

Prostate carcinoma cells, LNCaP and PC-3 cells or colon carcinoma cellsHT-29, were incubated with increasing concentration of the indicatedcompounds for 48 hours and cellular proliferation was measured using the³H-thymidine assay. The IC₅₀ of the positive control, doxorubicin, was0.02-0.08 μM in the cell lines used. The data shows that multiple cellslines such as PC-3 and HT29 do not cleave the above-indicated prodrugs.In contrast, an enzyme present in or on LNCaP cells cleaves several ofthe Ile containing analogs. The most potent analog is exemplified withSuc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:39) that has an IC₅₀ of 0.19 μM onLNCaP cells.

Example 3 Suc-Ile-Ala-Leu-Dox is Well-Tolerated in Healthy Mice

Suc-Ile-Ala-Leu-Dox, an exemplary tripeptide prodrug of the invention,is well tolerated in mice. In our initial single dose Maximum ToleratedDose (MTD) study, groups of five normal mice were administeredintravenous bolus doses of Suc-Ile-Ala-Leu-Dox. The mice were observeddaily for 28 days and body weights measured twice weekly. Dose levelstested were 0, 23, 47, 70, 93 and 117 mg/kg, equivalent to 0, 14, 28,42, 56, and 70 mg of doxorubicin/kg respectively. There was no acutetoxicity, and the only signs of toxicity observed was a decrease ingroup mean body weight for the highest dose group, which was lower thanthe vehicle control group throughout the study. However, there were nomortalities, and no animals exhibited morbidity. The 28-day single-doseMaximum Tolerated Dose (MTD) of Suc-Ile-Ala-Leu-Dox was not attained inthis experiment, and is therefore greater than the highest dose tested,117 mg/kg. This MTD (equivalent to 66 mg of doxorubicin/kg) is at least16-fold higher than that of doxorubicin alone, which results inmortality following doses greater than 4 mg/kg.

Post-analysis of compounds showed that they contained about 75% activecompound due to water content and impurity. So the doses mentioned inthe above paragraph overestimated amount of compound administered. Thesingle does MTD (equivalent to 70 mg of doxorubicin/kg) is at least3.3-fold higher than that of doxorubicin alone, which results inmortality following doses greater than 16 mg/kg.

The above-described experiment was re-performed (Example 8A) and thefollowing result was obtained: the single dose MTD ofSuc-Ile-Ala-Leu-Dox was determined to be 94 mg/kg (56 mg/kg doxorubicinequivalent), which is at least 3.5-fold higher than that of doxorubicinalone (16 mg/kg).

Example 4 Suc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:39) is Well Tolerated inHealthy Mice

Suc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:39), an exemplary tetrapeptideprodrug of the invention, is well tolerated in mice. In an initialsingle dose Maximum Tolerated Dose (MTD) study, groups of five normalmice were administered intravenous bolus doses ofSuc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:39). The mice were observed dailyfor 28 days and body weights measured twice weekly. Dose levels testedwere 0, 25, 50, 75, 100 and 125 mg/kg, equivalent to 0, 14, 28, 42, 56,and 70 mg of doxorubicin/kg respectively. No acute toxicity was observedfollowing administration. Toxicity, including paralysis and significantbody weight loss (>20% of their initial weight), was observed in the twohighest dose groups. On Day 14, four animals in the 125 mg/kg dose groupwere euthanized due to treatment-related toxicity. On Day 21, twoanimals in the next highest dose group, i.e., 100 mg/kg were similarlyeuthanized. There was no morbidity observed in the next dose-group (75mg/kg) and the group-mean-body-weight increased during the study.

Based on survival at Day 28, the single-dose MTD value forSuc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:39) was estimated to be 75 mg/kg(equivalent to a dose of 40 mg doxorubicin/kg). Thus, the MTD wasapproximately 10-fold higher than the MTD of doxorubicin alone,estimated to be 4 mg/kg based on the standard safe efficacious dose (4-8mg/kg). The above-described experiment was re-performed (Example 9).

Post-analysis of compounds showed that they contained about 75% activecompound due to water content and impurity. So the doses mentioned inthe above paragraph overestimated amount of compound administered. Thus,the single dose MTD of Suc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:39) wasapproximately 3.7-fold higher than that of doxorubicin alone (16 mg/kg).The more relevant comparison is the repeat-dose (RD) MTD, as a singledose is not efficacious. The RD-MTD of Suc-βAla-Ile-Ala-Leu-Dox (SEQ IDNO:39 was approximately 53 mg/kg, Q7Dx5 (30 mg/kg doxorubicinequivalent, based on efficacy study in Example 8, which was at least6.5-fold higher than the RD-MTD of doxorubicin (standard safe RDefficacious dose 4 mg/kg).

Example 5 Metabolism of Suc-Ile-Ala-Leu-Dox

The metabolism and clearance of Suc-Ile-Ala-Leu-Dox was studied innormal mice. The mice were administered Suc-Ile-Ala-Leu-Dox at a singleintravenous bolus dose of 117 mg/kg. Plasma samples were obtained at 1and 4 hours. Plasma samples of 100 μl were transferred to Eppendorftubes (1.5 mL) and an internal standard of daunorubicin (20 μL at 0.5mg/ml) was added together with acetonitrile (400 μl). The tubes werecapped and briefly vortexed followed by centrifugation at 14,000 rpm.420 μl from each tube was removed and dried in vacuo. Each sample wasreconstituted in 65 μl ammonium formate containing acetonitrile (20%)prior to analysis by reverse phase liquid chromatography in combinationwith tandem mass spectrometry (LC MS/MS).

Urine was collected at 2 and 24 hours post administration from pairs ofmice in metabolic cages. Urine samples were diluted with ammoniumformate containing acetonitrile (20%) to give a target analyteconcentration within the practical range of the LC MS/MS assay. 30 μl ofeach diluted sample was placed in an Eppendorf tube (1.5 ml) and aninternal standard of daunorubicin (20 μL at 0.5 mg/ml) was addedtogether with 50 μl of ammonium formate containing acetonitrile (20%).Each sample was then analyzed by LC MS/MS.

An Agilent HP 1100. HPLC with DAD detector and Chemstation software wascoupled to a PE Sciex API 365 mass spectrometer with an electrospray ionsource.

HPLC was performed on a TSK-Gel Super ODS, 2 mm, 4.6×50 mm (TosoHaas)reversed phase column equipped with a HAIGUARD C18 guard disc (HigginsAnalytical) and stainless steel frit (Upchurch Scientific).Chromatography was performed at room temperature. The flow rate was 0.5ml/min. Injection volume was 50 μl. Gradient elution was performed usinga mobile phase of 20 mM ammonium formate with increasing amounts ofacetonitrile. The API 365 was operated at 365° C. in a multiple reactionmonitoring mode, set to monitor specific analyte parent-daughter ionpairs. Integration of chromatograms was performed by MacQuan software(PE Sciex) and quantitation of each analyte obtained by comparison topreviously obtained calibration curves. Daunorubicin was used as aninternal standard in all cases.

As seen in Table 2 and FIG. 11, Suc-Ile-Ala-Leu-Dox was cleared from thecirculation very rapidly, with approximately 1.1% of the administereddose detected at 1 hour, while by 4 hours it was virtually undetectable.At 2 and 24 hours the parent compound was detected in urine at 3.9% and15.0% of the administered dose, showing that the kidney is a major organof excretion for the prodrug (FIG. 12).

TABLE 2 Plasma Urine 1 hr 4 hr 2 hr 24 hr Suc-Ile-Ala-Leu-Dox 1.14 0.003.92 15.0 Ala-Leu-Dox 0.00 0.00 0.00 0.00 Leu-Dox 0.02 0.00 1.17 2.25Dox 000 0.00 0.06 0.36

Percent of administered dose—plasma volume estimated at 40% of bloodvolume, which was calculated at 7% of animal's body weight.

Ala-Leu-Dox was not detected in plasma or urine. The major peptidemetabolite was Leu-Dox. Doxorubicin was virtually undetectable in plasmabut was found in urine at low levels at 2 and 24 hours. The levels ofboth parent and metabolites were higher in urine at 24 hours than at 2hours. This most likely resulted from later urination of the mice,relative to the initial urine collection time (2 hours). The urinevalues represent an accumulation from 0-2 hours and from 2-24 hours,thus later urination (after 2 hours) would be accumulated in the 2-24hour sample.

A low level of cleavage and activation of the Suc-Ile-Ala-Leu-Doxprodrug occurred in the blood of normal mice. The minimal toxicityobserved with Suc-Ile-Ala-Leu-Dox at the high dose-level tested (Example2), confirms that there is almost no systemic production of the activemetabolite doxorubicin which, when present systemically, is toxic tonormal tissues.

Example 6 Metabolism of Suc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:39)

The metabolism and clearance of Suc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:39)was studied in normal mice administered a single intravenous bolus doseat 117 mg/kg Suc-Ala-Ile-Ala-Leu-Dox (SEQ ID NO:39). Plasma samples wereobtained at 1 and 4 hours from separate mice. Urine was collected at 2and 24 hours post administration from pairs of mice in metabolic cages.The plasma and urine samples were prepared and analyzed as described inExample 4.

Suc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:39) was cleared from the circulationvery rapidly: approximately 1.0% of the administered dose could bedetected in plasma at 1 hour, while it was virtually undetectable at 4hours (Table 3 and FIG. 13). At 2 and 24 hours the urine contained 12.7%and 4.81% of the administered dose, indicating that the kidney is amajor organ of excretion for the prodrug (FIG. 14).

TABLE 3 Plasma* Urine* 1 hr 4 hr 2 hr 24 hr Suc-βAla-Ile-Ala-Leu-Dox1.03 0.00 12.7 4.81 (SEQ ID NO:39) Ala-Leu-Dox 0.00 0.00 0.01 0.00Leu-Dox 0.04 0.00 4.69 2.29 Dox 0.01 0.00 0.21 0.51 *Percent ofadministered dose

The metabolite Ala-Leu-Dox was virtually undetectable in plasma, and wasfound at very low levels in urine at 2. The major peptide metabolite wasLeu-Dox, which could be detected at low levels in plasma at 1 hour, aswell as in urine at 2 hours and 24 hours. Little free doxorubicin waspresent in plasma however comparatively high levels were detected inurine, showing that some complete metabolism is occurring.

In these samples, the levels of both the parent and metabolites werehigher in urine at 2 hours than at 24 hours. The sum of the amount ofSuc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:39) and metabolites collected at 0-2hours and 2-24 hours is similar to that of Suc-Ile-Ala-Leu-Dox (seeExample 4). Thus, there is no physiologically significant differencebetween the clearance of Suc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:39) andSuc-Ile-Ala-Leu-Dox.

Example 7 Comparative Metabolism in Mice

In a second study, three groups of ICR normal female mice wereadministered a single IV bolus dose with approximately 100 μmol/Kg ofSuc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:39), Suc-Ile-Ala-Leu-Dox or 10μmol/Kg of doxorubicin (Dox). Plasma was obtained from three individualanimals in each group at 5 minutes, 1, 2, 4, or 6 hr. Parent,dipeptidyl-doxorubicin (AL-Dox), α-aminoacyl-doxorubicin (L-Dox) anddoxorubicin concentrations were analyzed in extracts of the plasmasamples using a reverse phase gradient HPLC method with fluorescencedetection (λ_(ex)=480 nm, λ_(em)=560). Quantities were determined usinga linear standard curve fit to measurements of 10 to 2000 ng/mLdoxorubicin solutions in mouse plasma.

Concentration time courses indicate that metabolic patterns were similarfor both prodrug compounds. In particular, L-Dox was the majormetabolite over the first two hr while the dipeptidyl-conjugate AL-Doxwas a more minor product that formed at about the same time as L-Dox.Doxorubicin appeared later with the plasma concentration decreasing moreslowly over time than the other metabolites as expected from the currentand previously measured doxorubicin pharmacokinetic profiles and by thedoxorubicin control group. This is not consistent with sequentialcleavage profile from TOP/Trouase but is consistent with cleavage byanother enzyme of some type of endopeptidase activity at the next aminoacid. The activation is followed by sequential exopeptidase cleavage. Atwo fold decrease in exposure to doxorubicin is also observed with thetripeptide, Suc-Ile-Ala-Leu-Dox, compared to the correspondingtetrapeptide counterpart, Suc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:39). Itshould be noted that relative doxorubicin exposure after dosing thesecompounds is consistent with the relative safety observed in a maximumtolerated dose study in mice. Doxorubicin exposure was at least twicegreater when the corresponding compounds having leucine in place ofisoleucine was tested in the same experiment. This suggests that thehigh tolerance for the isoleucine compounds described in other examplescan be explained by lower doxorubicin exposure.

TABLE 4 Plasma exposure expressed as AUC_(0-6hr) of parent andpeptolytic metabolites following 100 μ mol/Kg single IV bolus injectionof indicated peptidyl-doxorubicin compound or 10 μ mol/Kg doxorubicin innormal mice. Parent AL-Dox L-Dox Dox Dosed Compound μM · hr (μM · hr)(μM · hr) (μM · hr) Suc-βAla-Ile-Ala-Leu- 326 0.5 8.5 1.5 Dox (SEQ IDNO:39) Suc-Ile-Ala-Leu-Dox 452 1.0 6.2 0.9 Doxorubicin N/A N/A N/A 3.3

Example 8 Suc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:39) is Well Tolerated inTumor Bearing Mice

The prodrug Suc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:39) therapeutic agent issignificantly better tolerated than doxorubicin under repeat-doseconditions. As would be expected, when a dose similar to the single-doseMTD (75 mg/kg) (See Example 4) was administered as a repeat-dose it wasless well tolerated. In repeat-dose studies in tumor bearing mice, threegroups of ten mice were dosed with 0, 53 or 68 mg/kgSuc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:39) (equivalent to 0, 30 and 38 mgdoxorubicin/kg), for a total five identical doses (Q5DX5) and observedfrequently for 60 days. All treated animals lost weight progressivelythroughout the study, while the vehicle control group gained up to 12%of the mean initial body weight. (FIG. 15). Two treated animals in thehigh dose group were terminated due to signs of toxicity, with bodyweight loss of greater that 20% of their initial weight. The signs oftoxicity were similar to those observed following a single, high dose(See Example 4) and were consistent with the known toxicity profile ofdoxorubicin in rodents. Thus cumulative toxicity resulted fromrepeat-dosing at these relatively high dose-levels. However the maximaloverall exposures to doxorubicin of the animals in the two dose groupsafter 5 doses were 150 and 190 mg/kg, respectively, which issignificantly higher (8 to 10 times) than the tolerated repeat doselevel of doxorubicin (4 mg/kg, or 20 mg/kg total exposure after 5doses). The RD-MTD of Suc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:39) wasapproximately 150 mg/kg doxorubicin equivalent which was at least6.5-fold higher than the tolerated repeat dose level of doxorubicin(standard safe RD efficacious dose 4 mg/kg, or 20 mg/kg total exposureafter 5 doses).

Example 9 Suc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:39) is Effective in TumorBearing Mice

The Suc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:39) therapeutic agent has provento be efficacious in extending the survival of mice and inhibiting thegrowth of human tumors in a mouse xenograft model utilizing thedoxorubicin-resistant colorectal carcinoma LS174t. For example, groupsof ten nude mice, subcutaneously implanted with chunks of LS174t whichwere allowed to grow to approximately 90 mg, were treated intravenouslywith 0, 53 or 68 mg/kg of Suc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:39)(equivalent to 0, 30 or 38 mg/kg doxorubicin) at five day intervals fora total of five identical doses (Q5DX5). Tumors and body weights weremeasured twice weekly for up to 60 days. As seen in FIG. 16, both doseswere efficacious in reducing the growth of tumors compared with vehiclecontrol animals. There was a dose-dependent increase in number of micesurviving to the Day 60 end-point of the study. There were 2 and 4long-term survivors in the low and high dose groups, respectively,compared with 0 in the vehicle control group. The Mean Day of Survival(MDS) in animals whose tumors reached 1.5 g prior to Day 60 wassignificantly better in the low (29.7 days) and high (23.4 day) dosegroups than in the vehicle control group (18.2 days). Thus,Suc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:39) was efficacious in thisaggressive human tumor model, in which doxorubicin alone at itstolerated dose (4 mg/kg), under this dosing regimen, is historicallyineffective.

Re-review of the results showed that some of the results wereincorrectly reported. There were 4 and 2 long-term survivors to the day60 end-point in the low and high dose groups, respectively, comparedwith 0 in the vehicle control group. Suc-βAla-Ile-Ala-Leu-Dox (SEQ IDNO:39) was efficacious in this aggressive human tumor model, in whichdoxorubicin alone at its tolerated dose (3 mg/kg), under this dosingregimen, is historically ineffective.

Example 10 Suc-Ile-Ala-Leu-Dox is Better Tolerated In Vivo thanDoxorubicin

Suc-Ile-Ala-Leu-Dox, an exemplary tripeptide prodrug of the invention,is well tolerated in mice. In a second single dose Maximum ToleratedDose (SD-MTD) study, groups of five normal ICR mice were administeredintravenously bolus doses of Suc-Ile-Ala-Leu-Dox. The mice were observeddaily for 49 days and body weights measured twice weekly. Dose levelstested were 0, 94, 117, 140 or 164 mg/kg, equivalent to 0, 56, 70, 84 or98 mg/kg of doxorubicin, respectively. There was no acute toxicity,within 24 hours, at any dose level. Dose and time dependent signs oftoxicity were observed during the study. Toxicity, including partialhind-end paralysis and significant body weight loss (>20% of theirinitial weight), was observed in the highest dose groups. Signs oftoxicity were partial hind end paralysis at 164 mg/kg by Day 14, weightloss at 140 mg/kg by Day 14 in one animal and at 117 mg/kg by Day 21also in one animal. Based on survival and lack of signs of toxicity atDay 49, the SD-MTD for Suc-Ile-Ala-Leu-Dox was determined to be 94 mg/kg(equivalent to 56 mg/kg of doxorubicin), which is 3.5-fold higher on amolar basis than the SD-MTD for doxorubicin alone (16 mg/kg). See TABLE5. This is an approximate SD-MTD determination based on a range of dosesat 14 mg/kg doxorubicin equivalents increments over the range tested.

TABLE 5 SD-MTD SD-MTD SD-MTD Molar Ratio Compound Name (mg/kg) (mg/kgDox=) (Dox=) Doxorubicin 16 16 1 Suc-Ile-Ala-Leu-Dox 94 58 3.5

Example 11 Suc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:39) is Better ToleratedIn Vivo than Doxorubicin

Suc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:39), an exemplary tetrapeptideprodrug of the invention, is well tolerated in mice. In a second singledose Maximum Tolerated Dose (SD-MTD) study, groups of five normal ICRmice were administered intravenous bolus doses ofSuc-βAla-Ile-Ala-Leu-Dox. The mice were observed daily for 49 days andbody weights measured twice weekly. Dose levels tested were 0, 50, 75 or100 mg/kg, equivalent to 0, 28, 42 or 56 mg/kg of doxorubicin,respectively. There was no acute toxicity, within 24 hours, at any doselevel. Dose and time dependent signs of toxicity were observed duringthe study. Toxicity, including partial hind-end paralysis andsignificant body weight loss (>20% of their initial weight), wasobserved in the highest dose group. By Day 21, three animals in the 100mg/kg dose were euthanized due to weight loss or paralysis. There was nomorbidity or mortality observed in the 75 mg/kg dose group. Based onsurvival and lack of signs of toxicity at Day 49, the SD-MTD forSuc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:39) was determined to be 75 mg/kg(equivalent to 42 mg/kg of doxorubicin), which is 2.6-fold higher on amolar basis than the SD-MTD for doxorubicin alone (16 mg/kg). See TABLE6 This is an approximate SD-MTD determination based on a range of dosesat 14 mg/kg doxorubicin equivalents increments over the range tested.

TABLE 6 SD-MTD SD-MTD SD-MTD (mg/kg Molar Ratio Compound Name (mg/kg)Dox =) (Dox =) Doxorubicin 16 16 1 Suc-β-Ala-Ile-Ala-Leu-Dox 75 42 26(SEQ ID NO:39)

Example 12 Advantages of Prodrugs Over the Unconjugated TherapeuticAgent

The prodrugs of the invention provide treatment advantages over thetherapeutic agent in its unconjugated form.

In the single dose Maximum Tolerated Dose (MTD) studies, groups ofnormal mice were administered intravenous bolus doses of the prodrug.The mice were observed daily for 28 days and body weights measured twiceweekly. The MTD was estimated to be equal to the highest dose thatproduced no death in mice after 28 days. As shown in Table 7, thesingle-dose MTD of the prodrugs range from 10-fold to at least 16-foldhigher than that of doxorubicin alone.

TABLE 7 Optimal Efficacy Optimal Repeat Efficacy Major SD MTD* Dose DosePlasma Compound (mg/kg) (mg/kg) Frequency MetaboliteSuc-βAla-Ile-Ala-Leu-Dox 75 (42) 68 (38) Q 5 days × 5 Leu-Dox (SEQ IDNO:39) Suc-Ile-Ala-Leu-Dox >117 (70) n.d. n.d. Leu-DoxSuc-βAla-Leu-Ala-Leu-Dox 75 (42) 71.2 (40) Q 1 week × 5 Ala-Leu-Dox (SEQID NO:74) Suc-Leu-Ala-Leu-Dox >117 (70) 63 (38) Q 5 days × 5 Leu-DoxDoxorubicin 4 (4) 4 (4) Q 1 week × 5 n.a. n.d. = not determined; n.a. =not applicable (values in parentheses are the molar equivalent dose ofdoxorubicin)

In repeat-dose studies in tumor bearing mice, groups of ten mice weredosed with various amounts of prodrug for a total of five doses ateither five day or 1 week intervals. After frequent observation over 60days, the dose which proved to be within acceptable toxicity limits andproduced an extension in survival was identified as the optimal efficacyrepeat dose. As seen in Table 7, optimal efficacy repeat dose of theprodrugs are approximately 9-fold higher than that of doxorubicin alone.Thus, the prodrugs permit a much greater amount of therapeutic agent tobe delivered to the body as a whole and thus to the vicinity of thetarget cell.

Post-analysis of compounds showed that they contained about 75% activecompound due to water content and impurity. So the doses mentioned inthe above paragraph overestimated amount of compound administered. Asshown in Table 8 which shows corrected results, the SD-MTD of theisoleucine containing prodrugs range from 1.6-fold to 2.5-fold higherthan that of doxorubicin alone. After frequent observation over 60 days,the dose which proved to be within acceptable toxicity limits wasidentified as the maximum tolerated repeat dose. As seen in Table 8,which shows corrected results RD-MTD of the prodrugs are approximately6.5-fold higher than that of doxorubicin alone. Repeat dosing of theprodrugs at or lower than their RD-MTD significantly prolong survival ofLS174t tumor bearing mice, whereas that of doxorubicin is completelyineffective. Thus, the conclusion remains the same in that the prodrugspermit a much greater amount of therapeutic agent to be delivered to thebody as a whole and to the vicinity of the target cell.

TABLE 8 SD MTD Repeat Dose Repeat Dose Major Plasma Compound (mg/kg)*MTD (mg/kg)* Frequency Metabolite Suc-βAla-Ile-Ala-Leu-Dox 75 (42) 53(30) Q 5D × 5 Leu-Dox (SEQ ID NO:39) Suc-Ile-Ala-Leu-Dox 94 (56) n.d.n.d. Leu-Dox Suc-βAla-Leu-Ala-Leu-Dox 50 (28) 57 (32) Q 7D × 5Ala-Leu-Dox (SEQ ID NO:74) Suc-Leu-Ala-Leu-Dox 59 (35) 52 (31) Q 5D × 5Leu-Dox Doxorubicin 16 (16) 4 (4) Q 7D × 5 n.a. n.d. = not determine;n.a. not applicable *: values in parentheses are the doxorubicinequivalent doseAnalytical Methods for the Remaining Examples

The peptide sequences, synthesized using either solid or solution phaseapproaches, were used without further purification if the analyticalHPLC (methods A, B & D) showed the crude product to be greater than 80%pure. If not, the material was purified using preparative HPLC Method C.

HPLC Method A

Analytical HPLC analyses were performed on a Waters 2690 using a C-18column (4 μm, 3.9×150 mm ID, flow rate 1 mL/min) eluting with a gradientof solvent A (0.1% TFA/H₂O) and solvent B (0.1% TFA/ACN) and the datawas processed at λ 254 nm using the Waters Millennium system. AnalyticalHPLC gradient started with 90% of solvent A and ended with 100% ofsolvent B over a period of 14 minutes (linear). Purity of the compoundsfor this method and the following ones was assessed as the relativepercentage area under the curve of the peaks.

HPLC Method B

Analytical HPLC analyses were performed on a Waters 2690 using a C-8column (3.5 μm, 4.6×150 mm ID, flow rate 1 mL/min) eluting with agradient of solvent A (80% 20 mM ammonium formate and 20% acetonitrile)and solvent B (20% 20 mM ammonium formate and 80% acetonitrile) and thedata was processed at λ 254 nm using the Waters Millennium system.Analytical HPLC gradient started with 100% of solvent A to 100% ofsolvent B over a period of 30 minutes (linear).

HPLC Method C

Preparative purification of crude products was achieved using a WatersDelta Prep 4000 system using a C-4 column (15 μm, 40×100 mm ID, flowrate 30 mL/min) eluting with a gradient of solvent A (H₂O), and solventB (MeOH). The preparatory HPLC gradient started with 80% of solvent Aand goes to 100% of solvent B over a period of 70 minutes (linear). UVdetection was at λ 254 nm.

HPLC Method D

Analytical HPLC was accomplished on a Hewlett Packard instrument using aTSK superODS column (TosoHaas); solvent A (TFA 0.1% in water); solvent B(TFA 0.1% in acetonitrile); gradient: 30 to 36% of B in 2 minutes, 36 to41% of B in 10 minutes, 41 to 90% of B in 3 minutes, 5 minutes at 90% B,detection wavelength λ 254 nm.

NMR and MS

Additional structural determinations were done by NMR and MS techniquesand the results supported the claimed compounds.

TLC Method

TLC analysis was carried out on silica gel 60F-254 nm-0.25 mm plates(Merck) with DCM/MeOH/H₂O/Formic acid 88% 85/15/1/2 for elution.

Ninhydrin Test

A few milligrams of product were introduced in a test tube, and twodrops of Solution A (50 mg/mL ninhydrin in ethanol), two drops ofSolution B (4 mg/mL phenol in ethanol), then two drops of Solution C (2mL 0.01M KSCN, aqueous in 100 mL pyridine) were added. The mixture wasleft in a boiling water bath for five minutes. In the presence of a freeamine the solution becomes purple.

Specific Oligopeptide Synthetic Examples Sources of CommerciallyAvailable Reagents

Doxorubicin and Daunorubicin were supplied by Meiji (Japan), Pd(PPh₃)₄by Strem chem (Newburyport, Mass.), PEG by Shearwater (Huntsville,Ala.), solvents, HATU by Aldrich (Milwaukee, Wis.); all resins and aminoacids were supplied by ABI (Foster City, Calif.), Novabiochem (SanDiego, Calif.), Advanced ChemTech (Louisville, Ky.), PeptideInternational (Louisville, Ky.), or SynPep (Dublin, Calif.).

Example 13 Synthesis of Fmoc-Ile-Ala-Leu-OH

Tripeptide (Fmoc-Ile-Ala-Leu-OH) was synthesized using solid-phaseapproach with standard Fmoc chemistry. A typical synthesis used Wang'salkoxy resin (0.60 mmol/μm loading). Fmoc-protected amino acids wereused for solid-phase peptide synthesis.

For a scale of 1 mM peptide on resin, 3 equivalents of amino acid waspreactivated with HBTU as the activating agent for 5 minutes beforebeing added to the resin together with 2 equivalents of DIEA. Thecoupling reaction was carried out for 2 h and then washed with DMF (25mL×3) and DCM (25 mL×3). The coupling reaction was repeated using 2equivalents of amino acid using similar conditions. The reactionprogress was monitored using ninhydrin test and if the ninhydrin testindicated incomplete reaction after 2 h then the coupling step wasrepeated for a third time. Deprotection was accomplished using 20%piperidine in DMF for 15-20 minutes. The coupling step was repeated withthe next amino acid until the desired peptide was assembled on resin.The final cleavage of peptide from the resin was accomplished bytreating the resin with a solution of 95% TFA and 5% water. Afterstirring the reaction mixture for 2 h at rt, the resin was filteredunder reduced pressure and washed twice with TFA. Filtrates werecombined and the peptide was precipitated by adding 400 mL of coldether. The peptide was filtered under reduced pressure and dried toyield Fmoc-Ile-Ala-Leu-OH (92% HPLC purity by method A). Crude peptidewas characterized by LC/MS and used for the next step without anyfurther purification.

Example 14 Synthesis of Fmoc-β-Ala-Ile-Ala-Leu-OH (SEQ ID NO:37)

Tetrapeptide (Fmoc-β-Ala-Ile-Ala-Leu-OH) (SEQ ID NO:37) was synthesizedusing solid-phase approach with standard Fmoc chemistry. A typicalsynthesis used Wang's alkoxy resin (0.60 mmol/μm loading).Fmoc-protected amino acids were used for solid-phase peptide synthesis.For a scale of 1 mM peptide on resin, 3 equivalents of amino acid waspreactivated with HBTU as the activating agent for 5 minutes beforebeing added to the resin together with 2 equivalents of DIEA. Thecoupling reaction was carried out for 2 h and then washed with DMF (25mL×3) and DCM (25 mL×3). The coupling reaction was repeated using 2equivalents of amino acid using similar conditions. The reactionprogress was monitored using ninhydrin test and if the ninhydrin testindicated incomplete reaction after 2 h then the coupling step wasrepeated for a third time. Deprotection was accomplished using 20%piperidine in DMF for 15-20 minutes. The coupling step was repeated withthe next amino acid until the desired peptide was assembled on resin.The final cleavage of peptide from the resin was accomplished bytreating the resin with a solution of 95% TFA and 5% water. Afterstirring the reaction mixture for 2 h at rt, the resin was filteredunder reduced pressure and washed twice with TFA. Filtrates werecombined and the peptide was precipitated by adding 400 mL of coldether. The peptide was filtered under reduced pressure and dried toyield Fmoc-β-Ala-Ile-Ala-Leu-OH (SEQ ID NO:37) (92% HPLC purity bymethod A). Crude peptide was characterized by MS and used for the nextstep without any further purification.

Example 15 Synthesis of Fmoc-Ile-Ala-Leu-Dox

Doxorubicin.HCl (2.34 g, 4.03 mmol) and Fmoc-Ile-Ala-Leu-OH (2.4 g, 4.48mmol) were dissolved at room temperature in anhydrous DMF (150 mL). Tothis rapidly stirred solution, DIEA (1.56 mL, 8.96 mmol) was added inone portion and the reaction mixture was stirred for 15 minutes at roomtemperature. The reaction mixture was cooled to 0° C. using an ice bathand 1.87 g (4.92 mmol) of HATU was added slowly over 10 minutes. Thereaction mixture was stirred for another 60 minutes at room temperature.Ice cold water (200 mL) was added to the reaction mixture, whichresulted in the formation of a red precipitate. The precipitate wascollected over a coarse frit, washed with 3×50 mL water and 3×50 diethylether and dried under reduced pressure to yield Fmoc-Ile-Ala-Leu-Dox(89% yield, 94% HPLC purity by method A). This product was characterizedby MS and used for the next step without any further purification.

Example 16 Synthesis of Fmoc-β-Ala-Ile-Ala-Leu-Dox (SEQ ID NO:75)

Doxorubicin.HCl (1.43 g, 2.5 mmol) and yield Fmoc-β-Ala-Ile-Ala-Leu-OH(SEQ ID NO:37) (1.6 g, 2.6 mmol) were dissolved at room temperature inanhydrous DMF (150 mL). To this rapidly stirred solution, DIEA (1 mL,5.7 mmol) was added in one portion and the reaction mixture was stirredfor 15 minutes at room temperature. The reaction mixture was cooled to0° C. using an ice bath and 1.07 g (2.8 mmol) of HATU was added slowlyover 10 minutes. The reaction mixture was stirred for another 60 minutesat room temperature. Ice cold water (200 mL) was added to the reactionmixture, which resulted in the formation of a red precipitate. Theprecipitate was collected over a coarse frit, washed with 3×50 mL waterand 3×50 diethyl ether and dried under reduced pressure to yieldFmoc-β-Ala-Ile-Ala-Leu-Dox (SEQ ID NO:75) (88% yield, 92% HPLC purity bymethod A). This product was characterized by MS and used for the nextstep without any further purification.

Example 17 Synthesis of Suc-Ile-Ala-Leu-Dox from Fmoc-Ile-Ala-Leu-Dox

To a solution of Fmoc-Ile-Ala-Leu-Dox (4.4 g, 4.13 mmol) in 20 mL of dryDMF, piperidine (20.4 mL, 206 mmol) was added in one portion resultingin a color change from red to purple. The reaction mixture was stirredfor 5 minutes at room temperature and then cooled to −20° C. using dryice/acetone bath. 21.2 g (210 mmol) of succinic anhydride was then addedto the cooled reaction mixture in one portion. The reaction was stirredrapidly at −5° C. for 5 minutes then at room temperature for another 90minutes. 750 mL of anhydrous diethyl ether was added to the reactionmixture, which resulted in the formation of a red precipitate. Thisprecipitate was isolated on a medium glass frit, washed with 2×50 mL ofdiethyl ether and dried under reduced pressure to yieldSuc-Ile-Ala-Leu-Dox (80% yield, 88% HPLC purity by method B). The finalproduct was purified using prep HPLC method C and characterized by LC/MSwhich gave a molecular weight of 939 (expected molecular weight 940).

Example 18 Synthesis of Suc-β-Ala-Ile-Ala-Leu-Dox (SEQ ID NO:39) fromFmoc-β-Ala-Ile-Ala-Leu-Dox (SEQ ID NO:75)

To a solution of Fmoc-β-Ala-Ile-Ala-Leu-Dox (SEQ ID NO:75) (4 g, 3.53mmol) in 40 mL of dry DMF, piperidine (17.4 mL, 176 mmol) was added inone portion resulting in a color change from red to purple. The reactionmixture was stirred for 5 minutes at room temperature and then cooled to−20° C. using dry ice/acetone bath. 18 g (180 mmol) of succinicanhydride was then added to the cooled reaction mixture in one portion.The reaction was stirred rapidly at −5° C. for 5 minutes then at roomtemperature for another 90 minutes. 750 mL of anhydrous diethyl etherwas added to the reaction mixture, which resulted in the formation of ared precipitate. This precipitate was isolated on a medium glass frit,washed with 2×50 mL of diethyl ether and dried under reduced pressure toyield Suc-β-Ala-Ile-Ala-Leu-Dox (SEQ ID NO:39) (78% yield, 86% HPLCpurity by method B). The final product was purified using prep HPLCmethod C and characterized by LC/MS which gave a molecular weight of1011 (expected molecular weight 1012).

Example 19 Synthesis of Fmoc-β-Ala-Ile-Ala-Leu-OBn (SEQ ID NO:76)

The Fmoc-β-Ala-Ile-Ala-Leu (SEQ ID NO:37), (24.34 g, 0.04 mol) is addedinto a round bottom flask with DMF (350 mL) and a magnetic stirrer.After the tetrapeptide is dissolved, benzyl bromide (4.76 mL, 0.04 mol),followed by cesium carbonate (13.04 g, 0.04 mol), is added to thesolution with stirring. The reaction mixture is stirred at roomtemperature for 1.5 hrs. Then, the reaction mixture is slowly pouredinto a flask with 450 mL of iced water. A large amount of white solidprecipitates out which is collected by suction filtration. The productis washed with water (2×200 mL) and placed in a vacuum desiccator.

Example 20 Synthesis of β-Ala-Ile-Ala-Leu-Obn (SEQ ID NO:77)

In a round bottom flask (25 mL), Fmoc-β-Ala-Ile-Ala-Leu-Obn (SEQ IDNO:76) (0.7 g, 1.0 mmol) is dissolved in 5 mL of anhydrous DMF.Piperidine (1.2 mL, 12.1 mmol) is added to the solution and the mixtureis stirred at room temperature for 25 minutes. The reaction is quenchedwith water (6 mL) and extracted with ethyl acetate (2×10 mL). Thecombined organic layer is further washed by water (2×5 mL), brine (5 mL)and dried over sodium sulfate. A white solid (0.8 g) is obtained afterremoval of solvent.

Example 21 Synthesis of MeOSuc-βAla-Ile-Ala-Leu-OBn (SEQ ID NO:78)

In a round bottom flask (250 mL), methyl hemisuccinate (3.19 g, 24.2mmol) is dissolved in anhydrous DMF (50 mL). DIEA (4.22 mL, 24.2 mmol)followed by HBTU (9.17 g, 24.2 mmol) are added into the solution. Themixture is stirred at room temperature for 45 minutes. To this mixtureis added a solution of βAla-Ile-Ala-Leu-Obn (SEQ ID NO:77) (crude,containing 10.14 g, 21.3 mmol) in anhydrous DMF (150 mL). The mixture iscontinually stirred at room temperature for 2.5 hrs. Then, the reactionmixture is slowly poured into a flask with 200 mL of iced water whilestirring. A large amount of white solid precipitates out which isextracted by ethyl acetate (3×200 mL). The combined organic layer isfurther washed by water (2×200 mL), brine (200 mL) and dried over sodiumsulfate.

Example 22 Synthesis of MeOSuc-βAla-Ile-Ala-Leu (SEQ ID NO:47)

MeOSuc-βAla-Ile-Ala-Leu-OBn (SEQ ID NO:78) (1.0 g; 1.46 mmol) is addedinto an Erlenmeyer flask with 100 mL of methanol. 50 mL of methanol isadded. The solution is transferred into a hydrogenation reaction vessel.To this vessel, Pd—C (90 mg, 10% wet, 50% water; 0.042 mmol) is added.After hydrogenation for 2 hours at room temperature, the reaction isstopped and the catalyst is filtered.

Example 23 Coupling of MeOSuc-βAla-Ile-Ala-Leu (SEQ ID NO:47) andDoxorubicin Using the “Urea Method”

Under dry nitrogen atmosphere, 26.04 g (52.0 mmol)MeOSuc-βAla-Ile-Ala-Leu (SEQ ID NO:47) and 23.26 g (40.2 mmol)doxorubicin hydrochloride are suspended/dissolved in 800 mL dry,urea-saturated (˜30% w/v) DMF and 19.948 mL DIEA. This mixture is cooledto 0-3° C. over ˜25 minutes. At this point 21.2 g (56.0 mmol) HATU isadded as a solution in ˜100 mL urea saturated DMF over 10 minutes (thevolume of this solution should be kept minimal). The reaction mixture isstirred for 10 minutes at −2 to 2° C. and poured into 4000 mL ice coldbrine, containing 2% v/v acetic acid over approximately five minuteswith vigorous stirring. The product is filtered off on a medium porosityfritted glass filter, washed generously with water and dried underreduced pressure.

Example 24 Synthesis of MeOSuc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:48)Therapeutic Agent

In a round bottom flask (50 mL), MeOSuc-βAla-Ile-Ala-Leu (SEQ ID NO:47)(0.25 g, 0.5 mmol) and doxorubicin (0.29 g, 0.5 mmol) are dissolved inanhydrous DMF (20 mL). After the mixture is stirred for 5 minutes, DIEA(0.17 mL, 1.0 mmol) followed by HBTU (0.19 g, 0.5 mmol) is added intothe solution. The mixture is stirred at room temperature for 4 hrs. DMFis removed by a rotary evaporator and the residue is taken up in 4.0 mL1:1 methylenechloride:methanol. To this solution, 40 mL of ether isslowly added while stirring. A precipitate is formed and collected bysuction filtration. The solid is washed with ether (2×10 mL) and driedin a vacuum desiccator.

Example 25 Removal of Free Doxorubicin from MeOSuc-β-Ala-Ile-Ala-Leu-Dox(SEQ ID NO:48)

MeOSuc-β-Ala-Ile-Ala-Leu-Dox (SEQ ID NO:48) (200 mg, 0.194 mmol), DIEA(0.068 mL, 0.388 mmol) and anhydrous DMF (10 mL) are placed in a 50 mlflask equipped with a magnetic stir bar. WhenMeOSuc-β-Ala-Ile-Ala-Leu-Dox (SEQ ID NO:48) had completely dissolved,isocyanate resin (390 mg, 0.582, pre-swollen in 5 mL of dichloromethanefor 5 minutes) is added and the resulting solution is stirred for 2 h atroom temperature with periodic HPLC monitoring. The reaction mixture isthen filtered through a frit to remove the resin when HPLC tracesindicate that the Dox is completely removed. The resin is washed with 10ml DMF and the DMF washes are combined with the filtered reactionmixture. The filtered reaction mixture washes are then concentrated on arotary evaporator equipped with a high vacuum pump and a 30° C. waterbath. The residue is suspended in 5 ml of DMF and the solution is thenslowly added into a rapidly stirred anhydrous diethylether solution. Theproduct is then filtered over a frit and washed with diethylether anddried under reduced pressure to give MeOSuc-β-Ala-Ile-Ala-Leu-Dox (SEQID NO:48).

Example 26 Hydrolysis of the MeOSuc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:48)Therapeutic Agent via Use of Cross Linked Enzyme

MeOSuc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:48) therapeutic agent (1.0 g,0.975 mmol) and 100 mL DMF are placed in a 500 mL flask. The suspensionis vigorously agitated with a magnetic stirrer. When theMeOSuc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:48) therapeutic agent hascompletely dissolved, 400 mL deionized water is added and the resultingsolution stirred at 35° C. A slurry of 1 g washed CLEC-PC (AltusBiologics) the immobilized enzyme is rinsed in three aliquots ofdeionized water then resuspended in 10 mL 20% aqueous DMF prior to use.The resulting suspension is stirred at 35° C. with periodic HPLCmonitoring. When all of the MeOSuc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:48)therapeutic agent has been consumed, the reaction mixture is filteredthrough a 0.45 μM nylon membrane filter to remove the CLEC-PC enzyme.The CLEC-PC cake is washed with 3×10 mL methanol and the methanol washesare combined with the filtered reaction mixture. The filtered reactionmixture plus methanol washes are then concentrated to a red gum on arotary evaporator equipped with a high vacuum pump and a 30° C. waterbath. The red gum is then suspended in 50 mL deionized water at roomtemperature and rapidly stirred via mechanical stirrer. To thissuspension a solution of 77.8 mg sodium bicarbonate (0.926 mmol, 0.95eq.) in 100 mL deionized water is added over 2 minutes. The suspensionis stirred at room temperature 20 minutes. The reaction mixture isfiltered through a 0.45 μM nylon membrane filter and lyophilized.

Example 27 Hydrolysis of the MeOSuc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:48)Therapeutic Agent Via Use of Soluble Enzyme

11.0 g (10.72 mmol) MeOSuc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:48)therapeutic agent is suspended in 800 mL HPLC-grade water andhomogenized for 60 minutes with an Ultraturrax T8 homogenizer to yield afinely divided suspension. This suspension is stirred (500 rpm) at 35°C. and adjusted to pH=6.05 with aq. 76 mM NaHCO₃. 1.0 g C. Antarctica“B” lipase (Altus Biologics) is then added and the reaction mixturestirred at 35° C. for 48 hours. During the 48 hr reaction time, pH ismaintained between 5.3 and 6.2 by periodic addition of 76 mM NaHCO₃ andthe reaction is periodically monitored by HPLC. After the reaction isnearly complete, the reaction mixture is then adjusted to pH=7 with aq.76 mM NaHCO₃ and filtered through a pad of Celite 521. The clarifiedreaction mixture is then acidified to ca. pH 3 with 5 mL glacial aceticacid. The precipitate is isolated by Celite 521 filtration, subsequentlyrinsing the Celite pad with methanol. The methanol solution is filteredthrough a 10-20 μM fritted glass filter and is dried by rotaryevaporation. This product is converted to the sodium salt by dissolutionin 70 mL 76 mM NaHCO₃ (0.95 eq.) and lyophilized. The product isidentical to that of Example 26.

Example 28 Immobilized Candida Antarctica “B” Lipase Hydrolysis ofMeOSuc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:48) Therapeutic Agent

30.0 g Candida Antarctica “B” lipase (Altus Biologics) is dissolved in300 mL water and dialyzed against 3×41 of 50 mM aq. NaHCO₃ (pH=6.4). 360mL of Pharmacia NHS-Activated Sepharose 4 Fast Flow is placed in acoarse glass fritted funnel and rinsed with 5×450 mL ice-cold 1 mM aq.HCl. The rinsed NHS-Activated Sepharose is then combined with thedialyzed enzyme solution. The resulting suspension is stirred at ambienttemperature (ca. 22° C.) for 2.0 hours. The Sepharose/enzyme conjugateis then isolated on a coarse fritted glass filter and then stirred in1000 mL 100 mM aq. TRIS (pH=7.45) for 15 minutes. This suspension isfiltered and incubated with another 1000 mL 100 mM aqueous TRIS buffer(pH=7.45) at 4° C., overnight. In the morning, the immobilized enzyme isfiltered off and after washing with water, is placed into a 2000 mLthree necked, round bottomed flask. 43 g MeOSuc-βAla-Ile-Ala-Leu-Dox(SEQ ID NO:48) therapeutic agent is added and the solids are suspendedin 800 mL deionized water. The flask is fitted with an overhead stirrer,and a pH-stat set to keep the pH of the reaction mixture between 5.9-6.2by controlling a syringe pump. The syringe pump is charged. 0.1 M NaHCO₃Progress of the reaction is followed by HPLC. After the reaction isnearly complete, the immobilized enzyme is filtered off and the liquidphase is lyophilized. The dry solids are then suspended in about 11 mLdry THF and filtered off.

Example 29 Large Scale Synthesis of Methyl Succinyl-N-Cap Form ofβAla-Ile-Ala-Leu-Dox (SEQ ID NO:40) Therapeutic Agent

120 mmol Doxorubicin.HCl and 199 mmol MeOSuc-βAla-Ile-Ala-Leu (SEQ IDNO:47) is dissolved in anhydrous DMF (10 L) under nitrogen. 76 mL DIEA(434 mmol) is added to the reaction mixture and the reaction mixture isstirred for 10 minutes at room temperature under nitrogen. The reactionmixture is then cooled to 0° C. over 10 minutes. In a separate flask asolution of 864 g HATU (220 mmol) in DMF (500 mL) is prepared. The HATUsolution is added slowly over 20 minutes to the reaction mixture whilethe reaction mixture is maintained at 0° C. The reaction mixture isstirred at 0° C. for 30 minutes.

A solution of NaCl (7.5 Kg, at least 30% w/v) in water (25 L) isprepared and cooled to 0° C. The reaction mixture is then slowly addedto the cooled brine solution with vigorous stirring over 120 minutes.The color of the solution must remain red, a blue solution indicatesthat the pH needs adjustment immediately to between 5.8-6.0 by addingacetic acid. The temperature is maintained at approximately 5° C. Thered precipitate is filtered off on a medium porosity fritted glassfilter, washed with water and dried under vacuum pressure over P₂O₅ toyield MeOSuc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:48).

Example 30 Treatment of MeOSuc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:48) withPs-isocyanate Beads to Remove Traces of Doxorubicin

146.4 g PS-isocyanate beads (240 mmol; supplied by Argonaut Lab, SanCarlos, Calif.) are dissolved in 1.5 L of anhydrous DMF and allowed toswell for 5-10 minutes at room temperature. The swelled beads arefiltered through a glass-fritted funnel and washed with additional 500mL of anhydrous DMF. 112 mmol MeOSuc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:48)is dissolved in 1000 mL of anhydrous DMF and 12 mmol mL DIEA is addedfollowed by the swelled PS-isocyanate beads. The reaction mixture isstirred at room temperature and is monitored using HPLC till the amountof doxorubicin peak is less than 0.1%. Analytical HPLC analyses areperformed using Water 2690 Column: Waters Symmetry Shield C₈ 3.5 μM4.6×150 mm (cat #WAT094269), solvent: A-80% aqueous 20 mM ammoniumformate (pH=4.5) 20% acetonitrile, solvent: B-20% aqueous 20 mM ammoniumformate (pH=4.5) 80% acetonitrile. Column temperature: controlled roomtemperature, sample Temperature 4° C., Run time: 37.5 minutes, detector:254 nm, Flow rate: 1.0 mL/min, Injection amount 10 μg (0.5 mg/mL×0.02mL), Mobile Phase A and B. Gradient: 37.5 minute linear gradient from100% mobile phase A to 100% mobile phase B with a 7.5 minuteequilibration delay.

When the doxorubicin peak is less than 0.1%, the reaction mixture isfiltered through a coarse sintered glass funnel to remove the beads. Abrine solution (at least 30% w/v) of 1.1 kg NaCl in 3.5 L water isprepared and cooled to 0° C. The filtered reaction mixture is thenslowly added to the cooled brine solution with vigorous stirring over 45minutes. The color of the solution must remain red, a blue solutionindicates that the pH needs adjustment immediately to between 5.8-6.0 byadding acetic acid. The red precipitate is filtered through a mediumsintered glass funnel, washed with water and dried under vacuum pressureover P₂O₅ to yield MeOSuc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:48) free ofany residual doxorubicin.

MeOSuc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:48) is dissolved in 1 L MeOH andthe methanol solution is then slowly added to 14 L of cooled ethyl etherwith vigorous stirring over 60 minutes. The red precipitate is filteredthrough a medium sintered glass funnel, washed with ether (1 L) anddried under vacuum pressure to yield MeOSuc-βAla-Ile-Ala-Leu-Dox (SEQ IDNO:48). The purity is determined by HPLC, as described in Example 29.

Example 31 Enzymatic Hydrolysis of MeOSuc-βAla-Ile-Ala-Leu-Dox (SEQ IDNO:48) to Yield Suc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:39)

The CLEC-CAB (Candida Antartica “B” Lipase) enzyme is purchased (fromAltus Biologics., Boston, Mass.) in solution form, where theconcentration of the enzyme is defined by the weight of dry enzyme permilliliter of solution. The crude enzyme suspension is shaken for fewminutes to obtain a homogenous solution. 504 mL (328 mmol) of thishomogenous solution is aliquoted into a flask. 2.5 L of deionized wateris added and the slurry is stirred for 10 minutes using a magneticstirrer. The enzyme solution is filtered using a coarse glass frittedfunnel, without taking the enzyme to dryness. The enzyme is transferredback into a flask. The enzyme is suspended in water and filtered threemore times.

The enzyme cake is resuspended into 550 mL of deionized water andtransferred into a RB flask. To this suspension,MeOSuc-βAla-Ile-Ala-Leu-Dox (SEQ ID NO:48) (106 mmol) is added and thereaction mixture is stirred at room temperature (25° C.). The pH of thereaction mixture is maintained between 5.8 and 6.1 by a pH-stat equippedwith a syringe pump charged with 1 N NaHCO₃ solution. Progress of thereaction is followed with periodic HPLC monitoring, as described inExample 29. The reaction is continued until the reaction seems to becomplete, as determined by HPLC.

To speed up the reaction, additional CLEC enzyme is required when thereaction is complete. Additional CLEC enzyme (homogenous solution) iswashed in a column format as described above. The enzyme cake isresuspended into 1.1 L of deionized water and added to the reactionmixture. The reaction mixture is stirred at room temperature withperiodic HPLC monitoring and the pH is maintained between 5.8 and 6.1.

Once the reaction is complete, the CLEC enzyme is removed from thereaction mixture by filtration through a 0.2 μM filter and rinsed with500 mL of deionized water. The filtrate is then lyophilized to yieldSuc-βAla-Ile-Ala-Leu-Dox.Na (SEQ ID NO:39).

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the appendedclaims.

1. In a method of making a prodrug, a method of removing freetherapeutic agent comprising: (1) coupling an optionally protectedstabilizing group-oligopeptide conjugate with the free therapeuticagent, (2) contacting the product of step (1) with a polymeric resin tobind free therapeutic agent remaining after step (1) and to form atherapeutic agent-polymeric resin complex, wherein the polymeric resinis polystyrene methylisocyanate, or polystyrene sulfonyl chloride, and(3) removing the therapeutic agent-polymeric resin complex.
 2. Themethod of claim 1 wherein the optionally protected stabilizinggroup-oligopeptide conjugate includes an oligopeptide of the formula(AA)_(n)-AA³-AA²-AA¹, wherein: each AA independently represents an aminoacid, n is 0 or 1, and when n is 1, then (AA)_(n) is AA⁴ whichrepresents any amino acid, AA³ represents isoleucine, AA² represents anyamino acid, and AA¹ represents any amino acid.
 3. The method of claim 1,wherein the therapeutic agent is selected from the group consisting ofDoxorubicin, Daunorubicin, Vinblastine, Vincristine, Calicheamicin,Etoposide, Etoposide phosphate, CC-1065, Duocarmycin, KW-2189,Methotrexate, Methopterin, Aminopterin, Dichloromethotrexate, Docetaxel,Paclitaxel, Epithiolone, Combretastatin, Combretastatin A4 Phosphate,Dolastatin 10, Dolastatin 11, Dolastatin 15, Topotecan, Camptothecin,Mitomycin C, Porfiromycin, 5-Fluorouracil, 6-Mercaptopurine,Fludarabine, Tamoxifen, Cytosine arabinoside, Adenosine Arabinoside,Colchicine, Cisplatin, Carboplatin, Bleomycin, Melphalan, chloroquine,and cyclosporin A.
 4. The method of claim 1, wherein the therapeuticagent is Daunorubicin or Doxorubicin.
 5. The method of claim 1, whereinthe therapeutic agent is Doxorubicin.
 6. In a method of making aprodrug, a method of removing free therapeutic agent comprising: (1)coupling an optionally protected stabilizing group-oligopeptideconjugate with the free therapeutic agent to produce a product mixturecomprising a prodrug of the therapeutic agent and remaining freetherapeutic agent, (2) contacting the product of step (1) with apolymeric resin having isocyanate or sulfonyl groups to bind freetherapeutic agent remaining after step (1) and to form a therapeuticagent-polymeric resin complex, wherein the polymeric resin is selectedfrom the group consisting of polystyrene isocyanate, polystyrenethioisocyanate, polystyrene methylthioisocyanate, polystyrenemethylsulfonyl chloride and polystyrene benzaldehyde; and (3) removingthe therapeutic agent-polymeric resin complex.
 7. The method of claim 6wherein the optionally protected stabilizing group-oligopeptideconjugate includes an oligopeptide of the formula (AA)_(n)-AA³-AA²-AA¹,wherein: each AA independently represents an amino acid, n is 0 or 1,and when n is 1, then (AA)_(n) is AA⁴ which represents any amino acid,AA³ represents isoleucine, AA² represents any amino acid, and AA¹represents any amino acid.
 8. The method of claim 6, wherein thetherapeutic agent is selected from the group consisting of Doxorubicin,Daunorubicin, Vinblastine, Vincristine, Calicheamicin, Etoposide,Etoposide phosphate, CC-1065, Duocarmycin, KW-2189, Methotrexate,Methopterin, Aminopterin, Dichloromethotrexate, Docetaxel, Paclitaxel,Epithiolone, Combretastatin, Combretastatin A4 Phosphate, Dolastatin 10,Dolastatin 11, Dolastatin 15, Topotecan, Camptothecin, Mitomycin C,Porfiromycin, 5-Fluorouracil, 6-Mercaptopurine, Fludarabine, Tamoxifen,Cytosine arabinoside, Adenosine Arabinoside, Colchicine, Cisplatin,Carboplatin, Bleomycin, Melphalan, chloroquine, and cyclosporin A. 9.The method of claim 6, wherein the therapeutic agent is Doxorubicin. 10.The method of claim 6, wherein the therapeutic agent is linked to theresin by a urea or sulfonamide group.